Method of producing recombinant proteins with mannose-terminated N-glycans

ABSTRACT

We describe a method of expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell, the method comprising: (a) introducing a nucleic acid encoding a recombinant protein into a Chinese Hamster Ovary (CHO) cell comprising a mutation in the GnT 1 gene (GenBank Accession Number AF343963) leading to loss of GnT 1 function; and (c) expressing the recombinant protein from the host cell, in which the expressed recombinant protein comprises a mannose-terminated glycan structure, and in which the method does not include a step of introducing functional GnT-I into the host cell. The method may be used for producing recombinant glucocerebrosidase with a mannose-terminated glycan structure, suitable for treatment or prevention of Gaucher&#39;s Disease.

This application is a divisional of U.S. patent application Ser. No.13/636,952, filed Oct. 19, 2012, now U.S. Pat. No. 9,175,278, issuedNov. 3, 2015, which is a U.S. National Stage of InternationalApplication No. PCT/SG2011/000118, filed Mar. 24, 2011, which claims thebenefit of U.S. Provisional Application No. 61/317,369, filed Mar. 25,2010, the entire content of each of which is hereby incorporated hereinby reference.

The foregoing applications, and each document cited or referenced ineach of the present and foregoing applications, including during theprosecution of each of the foregoing applications (“application andarticle cited documents”), and any manufacturer's instructions orcatalogues for any products cited or mentioned in each of the foregoingapplications and articles and in any of the application and articlecited documents, are hereby incorporated herein by reference.Furthermore, all documents cited in this text, and all documents citedor reference in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext or in any document hereby incorporated into this text, are herebyincorporated herein by reference. Documents incorporated by referenceinto this text or any teachings therein may be used in the practice ofthis invention. Documents incorporated by reference into this text arenot admitted to be prior art.

FIELD

This invention relates to the fields of biotechnology, molecular biologyand medicine. The invention in particular relates to a Chinese HamsterOvary cell line and its use in expression of recombinant proteins whichhave mannose-terminated N-glycans for targeting the protein to dendriticcells and macrophages, including glucocerebrosidase for the treatment ofGaucher disease, anti-cancer vaccines such as human mucin 1 (MUC1),HER2/neu and carcinoembryonic antigen (CEA) and anti-viral vaccines.

SEQUENCE LISTING

In accordance with 37 CFR §1.52 (e(5), a Sequence Listing in the form ofa text file (entitled “Sequence Listing_ST25.txt,” created on Feb. 11,2015, and 13 kilobytes in size) is incorporated herein by reference inits entirety.

BACKGROUND

Gaucher's disease is an autosomal recessive lysosomal storage diseasewhich results from a deficiency in the lysosomal enzyme,glucocerebrosidase. This causes the accumulation of glucoceramide withinperipheral macrophages, resulting in cellular enlargments. Gaucher'sdisease causes spleen, liver and bone marrow to malfunction anddeteriorate.

Ceredase is the commercial name for glucocerebrosidase derived fromhuman placenta. The intravenous infusion of Ceredase was shown to beclinically useful and has been approved for the treatment of Gaucher'sdisease in a study published in 1991 (Proc. Natl. Acad. Sci. 1990,1913-1916). In order for the enzyme to be taken up by the target celltype, the enzyme is first deglycosylated, resulting in amannose-terminated glycan structure so that it is taken up into themacrophage via its lectin receptors. This placental derivedglucocerebrosidase treatment was approved by the Federal DrugAdministration in April, 1991. However, due to the anticipated demandfor this new replacement enzyme therapy, ways to produce this proteinthrough means of recombinant expression have been sought.

Cerezyme is a recombinant form of glucocerebrosidase produced in CHOcells. The expressed protein is treated with exoglucosidases to producethe mannose sugars on the terminus of the existing polysaccharide chain,leading to a selective uptake of the enzyme by macrophages that arepresent in liver, spleen and skeleton. The drug works as well as thenaturally occurring protein does by catalyzing the hydrolysis of theglycolipid glucocerebroside to glucose and ceramide as part of thenormal degradation pathway for membrane lipids. However, the method ofproduction of Cerezyme involves extensive treatment with exoglucosidasesto trim the glycan structures to terminal mannose structures. Thisprocess is difficult to control and adds to the cost of production.

Major antigen presenting cells, such as dendritic cells and macrophages,express mannose-biding C-type lectins on their surface. Recombinantglycoproteins with mannose-terminated N-glycans are capable ofspecifically binding to and targeting such dendritic cells andmacrophages via the mannose-binding C-type lectin on macrophages.Accordingly, it is desired for recombinant proteins which are intendedfor use as vaccines to comprise mannose-terminated N-glycans. The sameproblems as described above for production of Cerezyme exist in relationto methods of production of other recombinant glycoproteins withmannose-terminated N-glycans for use, e.g., in vaccines.

There therefore exists a need in the art for a method of production ofglucocerebrosidase and other recombinant glycoproteins withmannose-terminated N-glycans which avoids such post-expressionprocessing.

One possibility is to make use of CHO cells which have mutations in thepost-translational glycosylation pathway. Van Patten et al. (2007,Glycobiology, 17, 467-478) describes a study carried out to examine theuse of alternative expression systems to produce Cerezyme (recombinantglucocerebrosidase) that would circumvent the need to useexoglucosidases to trim the glycan structures to terminal mannosestructures. The alternative expression systems tested includednon-mammalian expression systems, CHO cells cultured in kifunensine anda CHO glycosylation mutant, Lec 1.

The study showed that these different expression systems could indeedproduce glucocerebrosidase containing terminal mannose residues.Glucocerebrosidase produced by Lec 1 cells was in particular found tofunction just as well as the commercially available Cerezyme. However,the study also identified a number of significant issues with eachalternative.

With regard to use of Lec 1 cells, Van Patten et al. (2007,Glycobiology, 17, 467-478) found that both this expression system wascharacterised by low productivity. Given this, it would be difficult, ifnot impossible, to scale up expression to industrial production levels.

Furthermore, Lec 1 cells are not amenable to large scale culture. Lec 1cells were originally isolated from a proline auxotroph for geneticstudies and do not survive very well in normal large scale cell cultureconditions or in chemically defined media and cultured in suspension.Furthermore, the media has to supplemented with proline and even thenthe cells die easily. Thus this cell line is not robust and thereforenot suitable for use in the production of recombinant proteins as theirexpected growth characteristics and productivity is inferior to existingindustrial cell lines.

There therefore exists a need in the art for a scalable method ofproduction of glucocerebrosidase and other recombinant glycoproteinswith mannose-terminated N-glycans that avoid that avoids these problemsof the prior art.

SUMMARY

According to a 1^(st) aspect of the present invention, we provide amethod of expressing a recombinant protein comprising mannose-terminatedN-glycans from a host cell, the method comprising: (a) introducing anucleic acid encoding a recombinant protein into a Chinese Hamster Ovary(CHO) cell comprising a mutation in the GnT 1 gene (GenBank AccessionNumber AF343963) leading to loss of GnT 1 function; and (c) expressingthe recombinant protein from the host cell, in which the expressedrecombinant protein comprises a mannose-terminated glycan structure.

The method may be such that it does not include a step of introducingfunctional GnT-I into the host cell.

The method may be such that it does not include a step of introducing anucleic acid encoding functional GnT-I into the host cell.

The CHO cell may be selected with Ricinus communis agglutinin I (RCA-I)or a descendent thereof. The CHO cell selected with RCA-I or descendentthereof may comprise a mutation in the GnT-I gene.

The method may be such that selection with RCA-I comprises comprisingculturing CHO cells in the presence of Ricinus communis agglutinin I(RCA-I) and selecting cells which survive the culture, in which forexample: (a) the CHO cells are exposed to RCA-I at a concentration ofbetween 0.1 μg/ml to 100 μg/ml, for example up to 50 μg/ml or up to 20μg/ml, such as 10 μg/ml or 5 μg/ml; or (b) the CHO cells are exposedRCA-I for a period of from an hour, a few hours (such as 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12), overnight, to a few days, such as 2 days or 3days, such as overnight, optionally together with a further step ofselecting cells which do not react with RCA-I in an agglutination test.

The host cell may be adapted to suspension culture.

The host cell may comprise a JW152 cell line (deposited at ATCC underthe Budapest Treaty as accession number PTA-9657).

The method may be such that at step (b) an expression constructcomprising nucleic acid encoding the recombinant protein in anexpression vector such as pcDNA3.1 is introduced into the host cell.

The nucleic acid encoding the recombinant protein may be transformed orstably integrated into the host cell.

The method may be such that it further comprises the step of isolatingand/or purifying the expressed recombinant protein.

The protein may comprise (a) a sequence having GenBank Accession NumberNP_(—)000148.2 or a variant, homologue, derivative or fragment thereofhaving glucocerebrosidase activity; (b) a tumour-specific ortumour-associated antigen such as MUC1, HER2/neu and carcinoembryonicantigen (CEA) and antigenic portions thereof; or (c) a viral antigen.

The nucleic acid may comprise a sequence having GenBank Accession NumberNM_(—)000157 or a variant, homologue, derivative or fragment thereofencoding a protein comprising glucocerebrosidase activity.

There is provided, according to a 2^(nd) aspect of the presentinvention, a mannose-terminated recombinant protein such asglucocerebrosidase, MUC1, HER2/neu or carcinoembryonic antigen (CEA)expressed by a host cell line according to the 1^(st) aspect of theinvention.

We provide, according to a 3^(rd) aspect of the present invention, aJW152 cell (deposited at ATCC under the Budapest Treaty as accessionnumber PTA-9657) comprising a nucleic acid sequence encoding a proteinwith glucocerebrosidase activity, glucocerebrosidase, MUC1, HER2/neu orcarcinoembryonic antigen (CEA) and which is preferably capable ofexpression of such a protein.

As a 4^(th) aspect of the present invention, there is provided a methodof expressing a mannose-terminated protein having glucocerebrosidaseactivity from a host cell, glucocerebrosidase, MUC1, HER2/neu orcarcinoembryonic antigen (CEA), the method comprising: (a) providing aJW152 cell (deposited at ATCC under the Budapest Treaty as accessionnumber PTA-9657) comprising a nucleic acid encoding a protein havingglucocerebrosidase activity, glucocerebrosidase, MUC1, HER2/neu orcarcinoembryonic antigen (CEA); and (b) allowing the relevant protein tobe expressed from the cell.

We provide, according to a 5^(th) aspect of the present invention, amethod comprising expressing a mannose-terminated recombinant proteinfrom a host cell as set out above, or providing a mannose-terminatedrecombinant protein as set out above, and administering themannose-terminated recombinant protein to an individual in need thereof.

The method may be for treatment of Gaucher's Disease, in which themannose-terminated recombinant protein comprises a mannose-terminatedrecombinant protein with glucocerebrosidase activity.

The present invention, in a 6^(th) aspect, provides a mannose-terminatedrecombinant protein according to Claim 12 for use in a method oftreatment of Gaucher's Disease, in which the mannose-terminatedrecombinant protein comprises a mannose-terminated recombinant proteinwith glucocerebrosidase activity.

In a 7^(th) aspect of the present invention, there is provided a JW152cell (deposited at ATCC under the Budapest Treaty as accession numberPTA-9657) comprising a nucleic acid sequence encoding a protein, whichprotein when expressed is desired to comprise a mannose-terminatedglycan structure.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing showing the pathway by which glucosylceramide ishydrolysed by glucocerebrosidase into β-glucose and ceramide.

FIG. 2 is micrograph showing cells of a Gaucher patient, includingenlarged macrophages containing undigested glucocerebroside (fromwww.nlm.nih.gov/medlineplus/ency/imagepages/1450.htm).

FIG. 3 is a drawing showing a patient with Gaucher's disease (fromgeneticpeople.com/?p=276).

FIG. 4 is a drawing showing the structure of glucocerebrosidase.Glucocerebrosidase contains a single N-linked glycan attached to N-19.EMBO reports, 2003, 4:704.

FIG. 5 is a drawing showing that terminal mannose residues directCerezyme to target cells. Similarly, terminal mannose residues directglucocerebrosidase is produced in GnT I mutant CHO cells to targetcells.

FIG. 6 is a drawings showing protein N-Glycosylation in the ER and Golgiof mammalian cells. Helenius & Aebi, Science, 2001, 291:2364.

FIG. 7 is a drawing showing a comparison of N-glycosylation of EPO-FCproduced in CHO WT and JW152 cells.

FIG. 8 is a drawing showing a comparison of glucocerebrosidase activityin supernatant of transfected cells and untransfected cells.

SEQUENCE LISTING

SEQ ID NO: 1 is a nucleic acid sequence of aN-acetylglucoaminyltransferase I cDNA from CHO JW152 cells. SEQ ID NO: 2is an amino acid sequence of N-acetylglucoaminyltransferase I encoded bySEQ ID NO: 1.

DETAILED DESCRIPTION

Using a cytotoxic lectin, RCA-I, we have isolated a number of CHO mutantcell lines, including JW152, from CHO-K1 cells. Several of these stablytransfected lines have been adapted to suspension culture and grown inserum-free medium.

We demonstrate that recombinant proteins of interest, such asglucocerebrosidase, produced by such cells, including JW152 cells, thatare stably transfected with relevant expressing cDNAs comprisemannose-terminated glycan structures. These results suggest that JW152and other cells have the potential to become a host cell line forproducing proteins, such as mannose-terminated recombinant proteins (bywhich we mean recombinant proteins comprising mannose-terminatedN-glycans) including glycoprotein drugs and vaccines.

This is beneficial for expression of polypeptides which are to betargeted to cells with lectin receptors, for example, macrophages. Asdescribed in detail in this document, GnT-I deficient cells madeaccording to the methods described here may be used for the productionof recombinant glucocerebrosidase and other proteins of interest, whichhas mannose-terminated glycan structures and is hence able to be takenup by macrophages.

We therefore provide for a method of expressing a recombinant proteinfrom a host cell. The recombinant protein may compriseglucocerebrocidase, a viral antigen or a tumour antigen. The methodcomprises providing a host cell comprising a Chinese Hamster Ovary (CHO)cell which is selected with Ricinus communis agglutinin I (RCA-I) or adescendent thereof. The host cell may comprise JW152. The method maycomprise introducing a nucleic acid encoding a recombinant protein intothe host cell. Alternatively, or in addition, the host cell may alreadycomprise such a nucleic acid, prior to the process of selection. Thenucleic acid may be integrated into the genome of the host cell.

The method may further comprise the step of expressing the recombinantprotein from the host cell. The expressed recombinant protein maycomprise a mannose-terminated glycan structure. The method may be suchthat it does not include a step of introducing functional GnT-I into thehost cell.

Using such a method, the expressed recombinant protein may comprise oneor more N-glycan structures. We demonstrate that N-glycans produced inthese cells comprise mainly mannose-terminated structures. This is incontrast to the normal complex-type N-glycans that also contain sialicacid and galactose residues using expression from other cells.

Accordingly, we provide for the treatment or prevention of diseases suchas Gaucher's Disease, by such expression and administration ofmannose-terminated recombinant glucocerebrosidase, from such CHO hostcells.

We also provide for the treatment or prevention of diseases, by suchexpression and administration of mannose-terminated recombinant proteinsof interest, from such CHO host cells.

Such diseases include cancer, which according to the methods andcompositions described here may be treated or prevented by expressionand administration of mannose-terminated recombinant tumour-specific ortumour-associated antigens, from such CHO host cells. For example,anti-cancer vaccines may be made from MUC1, HER2/neu andcarcinoembryonic antigen (CEA)—or antigenic portions thereof—expressedaccording to the methods and compositions described here.

Examples of other tumour-specific and tumour-associated antigens areprovided in further detail below.

Further diseases include viral associated or caused diseases, whichaccording to the methods and compositions described here may be treatedor prevented by expression and administration of mannose-terminatedrecombinant viral antigens, from such CHO host cells. Examples of viralantigens are provided in further detail below.

In relation to Gaucher's Disease, mannose-terminated recombinantglucocerebrosidase protein for the treatment of disorders such asGaucher's Disease may be administered by intravenous infusion over 1-2hours. Dosage should be individualized to each patient. Initial dosagesmay range from about 2.5 U/kg of body weight 3 times a week to 60 U/kgonce every 2 weeks.

Disease severity may dictate that treatment be initiated at a relativelyhigh dose or relatively frequent administration. Dosage adjustmentsshould be made on an individual basis and may increase or decrease,based on achievement of therapeutic goals as assessed by routinecomprehensive evaluations of the patient's clinical manifestations.

For the purposes of the above paragraphs, an enzyme unit (U) is definedas the amount of enzyme that catalyzes the hydrolysis of 1 micromole ofthe synthetic substrate para-nitrophenyl-β-D-glucopyranoside (pNP-Glc)per minute at 37° C.

Use of such GnT-I deficient CHO cells as host cells has a number ofadvantages. First, as the recombinant expressed proteins of interesthave mannose-terminated glycan structures, there is no need forenzymatic treatment to expose these. Second, the GnT-I deficient CHOhost cells are derived from a parental CHO-K1 cell line that is veryrobust and are accordingly suited for recombinant expression inindustrial applications.

For example, JW152 cells originated from CHO-K1 cells grow very wellunder bioprocess conditions. Such JW152 cells have also been adapted tochemically defined medium and cultured in suspension.

The cell line JW152 was deposited on 11 Dec. 2008 at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, United States of America under the accession number PTA-9657as the International Deposition Number under the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

We therefore provide for a Chinese Hamster Ovary (CHO) cell or cell linewhich is capable of higher expression of mannose-terminated proteincompared to a wild type Chinese Hamster Ovary cell, and the use of sucha host cell or cell line for expression of mannose-terminatedrecombinant proteins.

The CHO cell capable of higher expression of mannose-terminated proteinmay be produced by a suitable selection method such as a RCA-1 selectionmethod. Such a method is described in further detail in “CHO Cells andCell Lines” below. Therefore, RCA-I can be used to isolate CHOglycosylation mutant cells that produce higher amounts ofmannose-terminated recombinant proteins.

Such a selection method is therefore specifically included in themethods and compositions described here.

We therefore provide for the use of a Chinese Hamster Ovary (CHO) cellor cell line which is capable of higher expression of mannose-terminatedprotein compared to a wild type Chinese Hamster Ovary cell, the CHO cellbeing obtainable by selection with Ricinus communis agglutinin I(RCA-I), in the expression of mannose-terminated recombinant proteins.In general, we provide a RCA-I resistant CHO cell or cell line, such asan RCA-I resistant CHO-K1 cell or cell line, for such a use.

Genetic analysis has revealed a dysfunctionalN-Acetyl-glucosaminyltransferase I (GnT I) gene in JW152 cells.Molecular cloning of the GnT I cDNA from the mutant cells identified apoint mutation that results in a premature stop codon. As a result, theJW152 cells can only synthesize a truncated version of GnT I proteinwith only 338 amino acids, rather than the normal protein which contains447 amino acids.

Using RCA-I we have isolated many more CHO mutant lines (about 100clones). Genetic analyses showed that they all lack functional GnT Igene. Many of them carry a different point mutation in the coding regionof GnT I gene, suggesting that they derived from different originalclones. Each of these may be used for expression of mannose-terminatedrecombinant proteins. The mutations in the GnT I genes from thesefurther CHO cell lines are shown in Table E1 below.

Accordingly, we provide for a Chinese Hamster Ovary (CHO) cell which iscapable of higher expression of mannose-terminated protein compared to awild type Chinese Hamster Ovary cell, the CHO cell comprising a mutationin the GnT I gene. We further provide for a Chinese Hamster Ovary (CHO)cell or cell line which is capable of higher expression ofmannose-terminated protein compared to a wild type Chinese Hamster Ovarycell, the CHO cell being obtainable by selection with Ricinus communisagglutinin I (RCA-I) and comprising a mutation in the GnT I gene. Weprovide for the use of such a cell or cell line as a host cell forexpression of recombinant proteins, such as mannose-terminatedrecombinant proteins

The mutation in the GnT I gene may comprise any point mutation,deletion, inversion, etc. The mutation in the GnT I gene may encode apartially functioning, or non-functioning GnT I polypeptide. Themutation in the GnT I gene may encode a truncated GnT I polypeptide.

We provide for CHO cells and cell lines comprising each of these mutantCHO cell lines and clones. We provide for specific cell lines derivedfrom RCA-I selection and capable of higher expression ofmannose-terminated protein compared to wild type or native CHO cells orparental cells. We provide for the use of such cells, host cells andcell lines for expression of mannose-terminated recombinant proteins.

We provide for mutant CHO nucleic acid and polypeptide sequencescomprised in such cells or cell lines, as described in further detailbelow.

The CHO cell or cell line may comprise a JW152 cell line. It maycomprise a JW80 cell line. It may comprise a JW36 cell line. It maycomprise a KFC15002 cell line. It may comprise a KFC15071 cell line. Itmay comprise a KFC5008 cell line. It may comprise a JW152 cell line. Itmay comprise a KFC5026 cell line. It may comprise a KFC20011 cell line.It may comprise a KFC15047 cell line.

The CHO cell or cell line may be transfected with a nucleic acidencoding a protein of interest, for example a heterologous orrecombinant protein. Such a protein might comprise a glycoprotein. Inparticular, such a protein might comprise a protein of interest such asa protein with glucocerebrosidase activity, a tumour-specific or tumourassociated antigen or a viral antigen, as described in further detailbelow.

As noted above, we provide for the nucleic acid themselves, e.g., anucleic acid encoding a mutant GnT I gene, or a fragment, variant,derivative or homologue of such a nucleic acid. The nucleic acidencoding a mutant GnT I gene, fragment, variant, derivative or homologuemay cause a CHO cell comprising it to be capable of higher expression ofmannose-terminated protein, compared to a CHO cell which does notcomprise such a nucleic acid, for example a wild type CHO cell. Thenucleic acid encoding the mutant GnT I gene may comprise a sequenceshown as SEQ ID NO: 1 or a variant, homologue, derivative or fragmentthereof.

GnT-I mutants made according to the methods described here show highlevels of sialylation, as described in PCT/SG2009/000348 (published as)in the presence of functional GnT-I. Accordingly, the CHO cell or cellline may be further transfected or co-transfected with a nucleic acidencoding a functional or full length or wild type GnT I sequence asdescribed in that document, should sialylation be desired.

SEQ ID NO: 1

The coding region of N-acetylglucoaminyltransferase I (Mgat1, GenBank:AF343963) mRNA isolated from the CHO JW152 cells. In these mutant cells,a C to T point mutation at position 1015 was identified (shown in bold):

ATGCTGAAGAAGCAGTCTGCAGGGCTTGTGCTTTGGGGTGCTATCCTCTTTGTGGGCTGGAATGCCCTGCTGCTCCTCTTCTTCTGGACACGCCCAGCCCCTGGCAGGCCCCCCTCAGATAGTGCTATCGATGATGACCCTGCCAGCCTCACCCGTGAGGTGTTCCGCCTGGCTGAGGACGCTGAGGTGGAGTTGGAGCGGCAGCGGGGGCTGTTGCAGCAAATCAGGGAGCATCATGCTTTGTGGAGACAGAGGTGGAAAGTGCCCACCGTGGCCCCTCCAGCCTGGCCCCGTGTGCCTGCGACCCCCTCACCAGCCGTGATCCCCATCCTGGTCATTGCCTGTGACCGCAGCACTGTCCGGCGCTGCTTGGATAAGTTGTTGCACTATCGGCCCTCAGCTGAGCATTTCCCCATCATTGTCAGCCAGGACTGCGGGCACGAAGAGACAGCACAGGTCATTGCTTCCTATGGCAGTGCAGTCACACACATCCGGCAGCCAGACCTGAGTAACATCGCTGTGCCCCCAGACCACCGCAAGTTCCAGGGTTACTACAAGATCGCCAGGCACTACCGCTGGGCACTGGGCCAGATCTTCAACAAGTTCAAGTTCCCAGCAGCTGTGGTAGTGGAGGACGATCTGGAGGTGGCACCAGACTTCTTTGAGTACTTCCAGGCCACCTACCCACTGCTGAGAACAGACCCCTCCCTTTGGTGTGTGTCTGCTTGGAATGACAATGGCAAGGAGCAGATGGTAGACTCAAGCAAACCTGAGCTGCTCTATCGAACAGACTTTTTTCCTGGCCTTGGCTGGCTGCTGATGGCTGAGCTGTGGACAGAGCTGGAGCCCAAGTGGCCCAAGGCCTTCTGGGATGACTGGATGCGCAGACCTGAGCAGCGGAAGGGGCGGGCCTGTATTCGTCCAGAAATTTCAAGAACGATGACCTTTGGCCGTAAGGGTGTGAGCCATGGGCAGTTCTTTGATCAGCATCTTAAGTTCATCAAGCTGAACCAGTAGTTCGTGTCTTTCACCCAGTTGGATTTGTCATACTTGCAGCGGGAGGCTTATGACCGGGATTTCCTTGCCCGTGTCTATAGTGCCCCCCTGCTACAGGTGGAGAAAGTGAGGACCAATGATCAGAAGGAGCTGGGGGAGGTGCGGGTACAGTACACTAGCAGAGACAGCTTCAAGGCCTTTGCTAAGGCCCTGGGTGTCATGGATGACCTCAAGTCTGGTGTCCCCAGAGCTGGCTACCGGGGCGTTGTCACTTTCCAGTTCAGGGGTCGACGTGTCCACCTGGCACCCCCACAAACCTGGGAAGGCTATGATCCTAGCTGGAATTAG

We further provide for mutant GnT I polypeptides, as well as fragments,variants, derivatives and homologoues thereof. The mutant GnT Ipolypeptide, fragment, variant, derivative or homologue may cause a CHOcell comprising it to be capable of higher expression ofmannose-terminated protein, compared to a CHO cell which does notcomprise such a polypeptide, for example a wild type CHO cell. Themutant GnT I polypeptide may comprise a sequence shown as SEQ ID NO: 2or a variant, homologue, derivative or fragment thereof.

SEQ ID NO: 2

The N-acetylglucoaminyltransferase I (GnT I) protein encoded by themutated gene in CHO JW152 cells. As a result of the point mutation(C1015T), JW152 cells only produce a truncated version of GnT I whichcontains only 338 amino acids rather than the normal protein thatcontains 447 amino acids. The C-terminal portion in bold is nottranslated in JW152 cells.

MLKKQSAGLVLWGAILFVGWNALLLLFFWTRPAPGRPPSDSAIDDDPASLTREVFRLAEDAEVELERQRGLLQQIREHHALWRQRWKVPTVAPPAWPRVPATPSPAVIPILVIACDRSTVRRCLDKLLHYRPSAEHFPIIVSQDCGHEETAQVIASYGSAVTHIRQPDLSNIAVPPDHRKFQGYYKIARHYRWALGQIFNKFKEPAAVVVEDDLEVAPDFFEYFQATYPLLRTDPSLWCVSAWNDNGKEQMVDSSKPELLYRTDFFPGLGWLLMAELWTELEPKWPKAFWDDWMRRPEQRKGRACIRPEISRTMTFGRKGVSHGQFFDQHLKFIKLNQQFVSFTQLDLSYLQREAYDRDFLARVYSAPLLQVEKVRTNDQKELGEVRVQYTSRDSFKAFAKALGVMDDLKSGVPRAGYRGVVT FQFRGRRVHLAPPQTWEGYDPSWN

Several of the stable lines have been adapted in suspension culture andgrown in serum-free medium. Proteins of interest such asglucocerebrosidase produced in serum-free medium comprisesmannose-terminated glycan structures. We therefore provide for the useof mutant CHO cells and cell lines derived from RCA-I selection, whichhave been adapted to suspension culture, or growth in semi-solid medium,in the production of mannose-terminated recombinant protein.

In conclusion, we have developed a novel method to express recombinantproteins with mannose-terminated glycan structures by use ofglycosylation mutant host cells isolated from CHO cells by RCA-Itreatment. All CHO cells that survive RCA-I treatment have very similarcharacteristics. First, they all lack a functional GnT I gene. Second,they express higher levels of mannose-terminated recombinant proteinsthan the wild type CHO cells. This feature remains the same both intransient transfection and in stably transfected cells.

The CHO glycosylation mutant cells isolated with RCA-I, such as JW152cells, can produce recombinant glycoproteins with high degree ofmannose-terminated glycan structures. This method can be used to producerecombinant glycoproteins in which presence of mannose-terminated glycanstructures is important for the efficacy. These proteins include forexample glucocerebrosidase, tumour-specific antigens, tumour-associatedantigens and viral antigens.

MUC1

MUC1 is also known as Mucin-1, MUC-1, Breast carcinoma-associatedantigen DF3, Carcinoma-associated mucin, Episialin, H23AG, PEMT,Peanut-reactive urinary mucin (PUM), Polymorphic epithelial mucin (PEM),Tumor-associated epithelial membrane antigen (EMA), Tumor-associatedmucin and CD227.

MUC1 has Swiss-Prot accession number P15941.3.

Representative nucleic acid sequences of MUC1 include: Human pancreaticmucin mRNA (GenBank Accession Number: J05582.1), Homo sapiens mucin 1,cell surface associated (MUC1), transcript variant 1, mRNA (NCBIReference Sequence: NM_(—)002456.4).

Representative amino acid sequences of MUC1 include: MUC1 (Homo sapiens)(GenBank Accession Number: CAA56734.1), mucin-1 isoform 1 precursor(Homo sapiens) (NCBI Reference Sequence: NP_(—)002447.4), mucin-1isoform 2 precursor (Homo sapiens) (NCBI Reference Sequence: NP_(—)001018016.1), mucin-1 isoform 3 precursor (Homo sapiens) (NCBI ReferenceSequence: NP_(—)001018017.1).

HER2/NEU

HER2 is also known as Receptor tyrosine-protein kinase erbB-2(EC=2.7.10.1). Other names include Metastatic lymph node gene 19 protein(MLN 19), Proto-oncogene Neu, Proto-oncogene c-ErbB-2, Tyrosinekinase-type cell surface receptor HER2, p185erbB2 and CD340.

HER2 has Swiss-Prot accession number P04626.

Representative nucleic acid sequences of HER2 include: Homo sapiensv-erb-b2 erythroblastic leukaemia viral oncogene homologue 2,neuro/glioblastoma derived oncogene homologue (avian)(ERBB2), transcriptvariant 1, mRNA (NCBI Reference Sequence: NM_(—)004448.2).

Representative amino acid sequences of HER2 include: Receptortyrosine-protein kinase erbB-2 isoform a (Homo sapiens) (NCBI ReferenceSequence: NP_(—)004439.2).

Carcinoembryonic Antigen (CEA)

Carcinoembryonic antigen (CEA) may include any of the following:

carcinoembryonic antigen-related cell adhesion molecule 5 preproprotein[Homo sapiens] (NCBI Reference Sequence: NP_(—)004354.2);

carcinoembryonic antigen-related cell adhesion molecule 1 isoform 1precursor [Homo sapiens] (NCBI Reference Sequence: NP_(—)001703.2);

carcinoembryonic antigen-related cell adhesion molecule 1 isoform 2precursor [Homo sapiens] (NCBI Reference Sequence: NP_(—)001020083.1)

carcinoembryonic antigen-related cell adhesion molecule 6 precursor[Homo sapiens] (NCBI Reference Sequence: NP_(—)002474.3)

carcinoembryonic antigen-related cell adhesion molecule 1 isoform 3precursor [Homo sapiens] (NCBI Reference Sequence: NP_(—)001171744.1)

carcinoembryonic antigen-related cell adhesion molecule 1 isoform 4precursor [Homo sapiens] (NCBI Reference Sequence: NP_(—)001171742.1)

carcinoembryonic antigen-related cell adhesion molecule 1 isoform 5precursor [Homo sapiens] (NCBI Reference Sequence: NP_(—)001171745.1)

carcinoembryonic antigen-related cell adhesion molecule 8 precursor[Homo sapiens] (NCBI Reference Sequence: NP_(—)001807.2)

pregnancy-specific beta-1-glycoprotein 5 precursor [Homo sapiens] (NCBIReference Sequence: NP_(—)002772.3)

CHO Cells and Cell Lines

The CHO cells and cell lines described here may be made by any suitablemeans. For example, the CHO cells and cell lines may be produced byselection using a suitable agglutinating agent, such as Ricinus communisagglutinin I (RCA-I).

We therefore provide a method of providing a CHO cell or cell line, themethod comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I) and selecting cells which survive theculture.

The CHO cells and cell lines described here may be made by treating astarting or parent cell with Ricinus communis agglutinin I (RCA-I) andselecting cells that survive such treatment. Such surviving cells may befurther cloned and made into cell lines. The selected cells and celllines may comprise higher expression of mannose-terminated protein asdescribed in this document. The selected cells and cell lines maycomprise mutant GnT I genes and polypeptides, as described in thisdocument.

For example, the CHO cells or cell lines described here may be selectedby exposing a parent cell line to Ricinus communis agglutinin I (RCA-I)at a suitable concentration for a suitable period.

The RCA-1 concentration could range from between 0.1 μg/ml to 100 μg/ml,for example up to 50 μg/ml or up to 20 μg/ml. Examples of specificconcentrations include 10 μg/ml and 5 μg/ml.

The period of incubation or exposure to RCA-1 could be from an hour, afew hours (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), overnight, to afew days, such as 2 days or 3 days.

In general, the period and concentration can be adjusted to eliminatethe majority of CHO cells, but to enable a small proportion of cells,which are resistant to RCA-I, to survive and form colonies. Within this,the concentration and period of exposure and selection may be varied,but generally, the higher the concentration of RCA-I, the lower theperiod of exposure is necessary, and vice versa.

The selection could be done on any suitable starting cell or cell line,but this will generally be a CHO cell or cell line. Any known CHO cellor cell line could be used as a starting point or parent cell, includingCHO-K1. Other suitable starting cells could include, but are not limitedto the following (ECACC accession numbers in brackets), CHO (85050302),CHO (PROTEIN FREE) (00102307), CHO-K1 (85051005), CHO-K1/SF (93061607),CHO/dhFr- (94060607), CHO/dhFr-AC-free (05011002), RR-CHOKI (92052129).A starting cell or cell line may also include CHO DG44 (Catalogue numberA11000-01, Invitrogen, Carlsbad, Calif., United States of America).

Following selection, the surviving cells are allowed to grow and formcolonies following which they may be picked. The time allowed for thiswill vary, but will generally be long enough for colonies to grow to apickable size. Examples of such times are 5 days, 7 days, 9 days, 11days, 13 days, one week, two weeks, three weeks or more.

The picking may be done manually, or it may be automated through use ofrobots, such as CLONEPIX (Genetix, New Milton, Hampshire, UK). Thepicked colonies may be further cloned, further screened, characterisedand cultured, etc.

The selected cells may be subjected to further tests. For example, theymay be subjected to agglutination tests using RCA-I to confirm themutant cells no longer react with RCA-I.

As a specific example, which is not intended to be limiting, CHO-K1cells may be cultured, for example in 6-well plates, to confluence.Culture media may be changed to serum-free DMEM. Ricinus communisagglutinin I (RCA-I, EY Laboratories, San Mateo, Calif., United Statesof America) may be added into the media to reach a final concentrationof 10 μg/gml. This may be incubated with cells overnight. The serum-freeDMEM containing RCA-I may be replaced, for example with fresh DMEM with10% FBS. Nine days later, colonies of the CHO cells that survive theRCA-I treatment may be picked and cultured, for example in 24 wellplates.

These cells may be subjected to further tests, such as agglutinationtests using RCA-I to confirm the mutant cells no longer react withRCA-I. We therefore provide for a method of providing a CHO cell or cellline, the method comprising culturing CHO cells in the presence ofRicinus communis agglutinin I (RCA-I), selecting cells which survive theculture and which do not react with RCA-I in an agglutination test.

The RCA-I selected CHO cells and CHO cell lines may be tested for theirability to produce mannose-terminated glycan structures on expressedproteins, by for example expressing a protein of interest anddetermining the amount of mannose-terminated glycan structures on theprotein. This may be done by the methods known in the art. One exampleof such a method is the “Assay for Protein-Derived N-LinkedOligosaccharides”, described in detail below.

We therefore provide for a method of providing a CHO cell or cell line,the method comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I), selecting cells which survive the cultureand selecting those cells or cell lines which display higher expressionof mannose-terminated proteins, and using the resulting cells or celllines as host cells for expression of mannose-terminated recombinantproteins.

The GnT I gene in such selected cells may be cloned and sequenced, usingmethods known in the art. The GnT I gene may comprise a mutant GnT Igene as described here.

We therefore provide for a method of providing a CHO cell or cell line,the method comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I), selecting cells which survive the cultureand selecting those cells or cell lines which comprise mutant GnT Igenes as described herein. The method may comprise a further step ofexpressing a recombinant protein from the CHO cell or cell line.

Assay for Protein-Derived N-Linked Oligosaccharides

The monosaccharide composition of purified expressed protein ischaracterized by modified high-performance anion exchangechromatography; monosaccharides are released from an aliquot of proteinby heating with 4 M trifluoroacetic acid at 100 8C for 2 h and driedunder a vacuum.

The monosaccharides reconstituted in sterile distilled water areanalyzed using a waveform and DX500 system (DIONEX, Sunnyvale, Calif.).A CarboPac PA-1 column (DIONEX) is used to resolve monosaccharides in 18mM sodium hydroxide solution with a flow rate of 0.8 mL/min at 35° C. asdescribed previously (Shinkawa et al. 2003).

The oligosaccharide profile of each purified protein is characterized bymodified matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) with a positive ion mode as describedpreviously (Papac et al. 1998); N-linked oligosaccharides are releasedfrom 30 mg of IgG1 by incubation with lunit of recombinantpeptide-N-glycosidase F (PNGaseF; Sigma-Aldrich) for 18 h at 378C in 10mM Tris-acetate (pH 8.3).

The released oligo-saccharides are recovered after precipitation of theprotein with 75% ethanol. Following drying of the recovered supernatant,the oligosaccharides are dissolved in 13 mM acetic acid and incubated atroom temperature for 2 h. The acid-treated samples are desalted withcation-exchange resin (AG50W-X8, hydrogen form; BioRad, Hercules,Calif.) and dried in a vacuum.

The dried samples are dissolved in deionized water and mixed with thematrix super-DHB solution (Bruker Daltonics) to be characterized by aMALDI-TOF MS spectrometer Reflex III (Bruker Daltonik GmbH, Bremen,Fahrenheitstr, Germany) equipped with delayed extraction.

All samples are irradiated with ultraviolet light (337 nm) from an N2laser; positive ions are acceler-ated to 20kV and analyzed in areflectron mode. The oligosaccharide standards (TAKARA BIO Inc., Shiga,Japan) are employed.

Mutant Cho Cells and Cell Lines

We provide for a CHO cell or cell line derived from RCA-I selection, asdescribed above. Such a cell line could include a JW152 cell line, orany of the cell lines set out in Table E1 below, including a JW80 cellline, a JW36 cell line, a KFC15002 cell line, a KFC15071 cell line, aKFC5008 cell line, a JW152 cell line, a KFC5026 cell line, a KFC20011cell line or a KFC15047 cell line.

Protein Expression

The CHO cells described here may be used as host cells for expression ofany protein of interest. This may be done by means known in the art.

Protein expression in CHO cells and cell lines is well described in theliterature, and the skilled person will have little difficulty in usingthe CHO cells and cell lines described here as hosts for proteinexpression. Thus, for example, the CHO cells and cell lines may betransfected by means known in the art with expression vectors capable ofexpressing the protein of interest.

The CHO cells and cell lines may further be capable of expressing wildtype or functional GnT I, for example, the sequence set out in GenBankaccession number AF343963. This may be done by transfecting the CHOcells and cell lines with an expression vector encoding GnT I. This maybe on the same or different vector as that which contains the nucleicacid encoding the protein of interest

Any suitable protein may be expressed using the CHO cells described hereas host cells. The protein may comprise a heterologous protein. Theprotein may comprise a recombinant protein. The protein may comprise anengineered protein. The protein may comprise a glycoprotein.

Examples include heterologous proteins of therapeutic or pharmacologicalinterest. Proteins which may be expressed include anti-EGFR mAb,α-glucosidase, laronidase, Ig-CTLA4 fusion,N-acetylgalactosamine-4-sulfatase, luteinizing hormone, anti-VEGF mAb,Factor VIII, anti-lgE mAb, anti-CD11a mAb, α-galactosidase,interferon-β, anti-TNFα mAb, erythropoietin, anti-CD52 mAb, Factor VIII,tissue plasminogen activator, anti-HER2 mAb, TNFα receptor fusion,Factor IX, follicle stimulating hormone, anti-CD20 mAb, interferon-β,β-glucocerebrosidase, deoxyribonuclease I, etc. Further examples includetumour-specific antigens, tumour-associated antigens and viral antigens,etc as described in further deail below.

In a specific example, we describe the expression of glucocerebrosidasefrom the CHO cells and cell lines described here.

Protein of Interest

The protein of interest which is expressed by the methods andcompositions described in this document may comprise any protein. Inparticular, such proteins comprise those for which presence ofmannose-terminated N-glycans is desired for whatever reason.

Examples of such proteins of interest include proteins for treatment ofspecific diseases, including glucocerebrosidase for the treatment ofGaucher disease. Other proteins of interest include proteins suitablefor use as anti-cancer vaccines such as human mucin 1 (MUC1), HER2/neuand carcinoembryonic antigen (CEA), as well as antigens for use invaccines for treatment or prophylaxis of various diseases.

As used herein, the term “antigen” refers to any substance that iscapable of being the target of an immune response. An antigen may be thetarget of, for example, a cell-mediated and/or humoral immune responseraised by a patient. The term “antigen” encompasses for example viralantigens, tumour-specific or -related antigens, bacterial antigens,parasitic antigens, allergens and the like.

Tumour Specific or Tumour Related Antigens (Anti-Cancer Vaccines)

The protein of interest may comprise a tumor-specific or tumour-relatedantigen, which may be suitable for use as an anti-cancer vaccine.

Tumor-specific or -related antigens include for example antigens frombreast cancer, colon cancer, rectal cancer, cancer of the head and neck,renal cancer, malignant melanoma, laryngeal cancer, ovarian cancer,cervical cancer, prostate cancer. Cancer antigens are antigens which canpotentially stimulate apparently tumor-specific immune responses. Someof these antigens are encoded, although not necessarily expressed, bynormal cells. These antigens can be characterized as those which arenormally silent (i.e., not expressed) in normal cells, those that areexpressed only at certain stages of differentiation and those that aretemporally expressed such as embryonic and fetal antigens. Other cancerantigens are encoded by mutant cellular genes, such as oncogenes (e.g.,activated ras oncogene), suppressor genes (e.g., mutant p53), fusionproteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried on RNA and DNA tumor viruses. Somenon-limiting examples of tumor-specific or -related antigens includeMART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-AL MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family (e.g. MUC-1), HER2/neu, p21ras, RCAS1,alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin andgamma-catenin, p120ctn, gp100^(Pmelll7) PRAME, NY-ESO-1, cdc27,adenomatous polyposis coli protein (APC), fodrin, Connexin 37,Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such ashuman papilloma virus proteins, Smad family of tumor antigens, lmp-1,P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase,SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, andc-erbB-2.

For example, the methods and compositions described here may be used toexpress cancer antigens such as human mucin 1 (MUC1), HER2/neu orcarcinoembryonic antigen (CEA), or antigenic parts thereof, inrecombinant forms which comprise mannose-terminated N-glycans fortargeting the protein to dendritic cells and macrophages, such formsbeing suitable for use as or in anti-cancer vaccines.

Viral Antigens

The protein of interest may comprise a viral antigen.

Viral antigens include for example antigens from hepatitis viruses A, B,C, D & E, HIV, herpes viruses, cytomegalovirus, varicella zoster,papilloma viruses, Epstein Barr virus, influenza viruses, para-influenzaviruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses,respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus,papovirus, mumps virus, measles virus; some non-limiting examples ofknown viral antigens include the following: antigens derived from HIV-1such as tat, nef, gp120 or gp160, gp40, p24, gag, env, vif, vpr, vpu,rev or part and/or combinations thereof; antigens derived from humanherpes viruses such as gH, gL gM gB gC gK gE or gD or or part and/orcombinations thereof or Immediate Early protein such asICP27, ICP47,ICP4, ICP36 from HSV1 or HSV2; antigens derived from cytomegalovirus,especially human cytomegalovirus such as gB or derivatives thereof;antigens derived from Epstein Barr virus such as gp350 or derivativesthereof; antigens derived from Varicella Zoster Virus such asgp1, 11,111 and IE63; antigens derived from a hepatitis virus such as hepatitisB, hepatitis C or hepatitis E virus antigen (e.g. env protein E1 or E2,core protein, NS2, NS3, NS4a, NS4b, NSSa, NSSb, p7, or part and/orcombinations thereof of HCV); antigens derived from human papillomaviruses (for example HPV6,11,16,18, e.g. L1, L2, E1, E2, E3, E4, E5, E6,E7, or part and/or combinations thereof); antigens derived from otherviral pathogens, such as Respiratory Syncytial virus (e.g F and Gproteins or derivatives thereof), parainfluenza virus, measles virus,mumps virus, flaviviruses (e. g. Yellow Fever Virus, Dengue Virus,Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenzavirus cells (e.g. HA, NP, NA, or M proteins, or part and/or combinationsthereof).

Representative viral antigen sequences include the following:

Envelope glycoprotein B [Human herpesvirus 5 (HHV-5)] (>giI219879660IgbIACL51135.11 envelope glycoprotein B [Human herpesvirus 5); Humanherpesvirus 5 strain AD169 substrain varUC, complete genome (GenBank:FJ527563.1); Human herpesvirus 4 (Epstein-Barr virus) glycoprotein350/220 [Human herpesvirus 4 (Epstein-Barr virus)] (GenBank:ADN85307.1), Human herpesvirus 4 isolate T41 glycoprotein 350/220(BLLF1) gene, partial cds (GenBank: HM366438.1).

Bacterial Antigens

The protein of interest may comprise a bacterial antigen, which may besuitable for use as an anti-bacterial vaccine.

Bacterial antigens include for example antigens from Mycobacteriacausing TB and leprosy, pneumocci, aerobic gram negative bacilli,mycoplasma, staphyloccocal infections, streptococcal infections,salmonellae, chlamydiae, neisseriae.

Other Antigens

The protein of interest may comprise other antigens. Other antigensinclude for example antigens from malaria, leishmaniasis,trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis, etc.

Glucocerebrosidase Sequences

We disclose the use of glucocerebrosidase sequences at both amino acidand nucleic acid level, for the expression of glucocerebrosidase withhigh mannose-termination in the CHO cells anc cell lines and host cellsdescribed here.

Example glucocerebrosidase amino acid sequences include GenBankAccession Number NP_(—)000148.2 while example glucocerebrosidase nucleicacid sequences include GenBank Accession Number NM_(—)000157.

Mutant Gnt I Sequences

We disclose mutant GnT I sequences comprising mutant GnT I amino acidsequences and mutant GnT1 nucleic acid sequences. Example mutant GnT Iamino acid sequences include the sequence shown as SEQ ID NO: 2, as wellas the sequences comprising the mutations shown in column 3 of Table E1below. Example mutant GnT I nucleic acid sequences include the sequenceshown as SEQ ID NO: 1, as well as the sequences comprising the mutationsshown in column 2 of Table E1 below. Table E1 shows the mutations in theGnT I sequence of clones isolated from selection with RCA-I, asdescribed in the Examples below.

Corresponding mutations are tabulated alongside showing respectivenucleotide and amino acid mutation and possible location of thedisruption in secondary structure/interaction or the resulting loss inamino acids in the case of a stop codon mutation. A minimum of 4bacteria colonies were sequenced to ensure that the mutations found werenot due to PCR error.

Mutant Gnt I Polypeptides and Glucocerebrosidase Polypeptides

The CHO cells and cell lines comprise mutant GnT I polypeptides. Theymay be used to express proteins or polypeptides of interest, such asglucocerebrosidase polypeptides.

We therefore provide generally for a mutant GnT I polypeptide, a proteinor polypeptide of interest such as a glucocerebrosidase polypeptide,together with fragments, homologues, variants and derivatives thereof.These polypeptide sequences may comprise the polypeptide sequencesdisclosed here, and particularly in the sequence listings.

A mutant GnT I polypeptide or protein of interest such as aglucocerebrosidase polypeptide (as the case may be) may comprise one ormore changes compared to the wild type GnT I or wild type protein ofinterest such as a glucocerebrosidase sequence. In the case oof GnT I,mutations may result from stop codons being introduced in the encodingnucleic acid sequence and consequent premature termination oftranslation of the GnT I mRNA.

The mutant GnT I polypeptide may be shorter than a wild type GnT Ipolypeptide. It may be a truncated version of wild type GnT Ipolypeptide. The length of the mutant GnT I polypeptide may be 90% orless, 80% or less, 70% or less, etc than the wild type sequence.

Similarly, the polypeptide of interest may be shorter than a wild typepolypeptide of interest. It may be a truncated version of wild typepolypeptide of interest. The length of the polypeptide of interest maybe 90% or less, 80% or less, 70% or less, etc than the wild typesequence.

For example, a polypeptide of interest or a mutant GnT I polypeptide maybe missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or more C-terminal residues compared to full lengthor wild type GnT I or polypeptide of interest polypeptide.

The mutant GnT I polypeptide may for example comprise a sequence set outin SEQ ID NO: 2. This is the mutant GnT I polypeptide sequence from thecell line JW152. The mutant GnT I polypeptide may comprise a GnT Isequence comprising a mutation set out at column 3 of Table E1 below.

The polypeptide of interest such as a glucocerebrosidase polypeptide mayfor example comprise a sequence shown as GenBank Accession NumberNP_(—)000148.2.

It will be understood that the mutant GnT I and polypeptide of interestsuch as glucocerebrosidase polypeptide sequences disclosed here are notlimited to the particular sequences set forth in the sequence listing,or fragments thereof, or sequences obtained from polypeptide of interestsuch as glucocerebrosidase protein or a mutant GnT I protein, but alsoinclude homologous sequences obtained from any source, for examplerelated cellular homologues, homologues from other species and variantsor derivatives thereof, provided that they have at least one of thebiological activities of mutant GnT I or polypeptide of interest such asglucocerebrosidase, as the case may be.

This disclosure therefore encompasses variants, homologues orderivatives of the amino acid sequences set forth in the sequencelistings, as well as variants, homologues or derivatives of the aminoacid sequences encoded by the nucleotide sequences disclosed here. Whererelevant to GnT I, such a sequence is generally referred to as a “mutantGnT I sequence” and where relevant to polypeptides of interest such asglucocerebrosidase, such a sequence is generally referred to as a“polypeptide sequence” or “protein sequence” or “glucocerebrosidasesequence”.

The length of the polypeptide of interest such as glucocerebrosidase ormutant GnT I polypeptide may be 90% or less, 80% or less, 70% or less,etc than a corresponding wild type sequence.

For example, a mutant GnT I nucleic acid may encode a mutant GnT Ipolypeptide that is missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100 or more C-terminal residues.Similarly, a polypeptide of interest such as glucocerebrosidase nucleicacid may encode a polypeptide of interest such as glucocerebrosidasepolypeptide that is missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100 or more C-terminal residues.

Biological Activities

In some embodiments, the sequences comprise at least one biologicalactivity of a polypeptide of interest such as glucocerebrosidase ormutant GnT I, as the case may be. As relevant to GnT I, the biologicalactivity may comprise improved ability to express proteins with highermannose-terminated glycan residues compared to wild type GnT I.

As relevant to glucocerebrosidase, the biological activity may compriseability to catalyse the hydrolysis of the glycolipid glucocerebroside toglucose and ceramide. In general, the term “glucocerebrosidase” shouldtherefore be read as encompassing any polypeptide havingglucocerebrosidase activity, including full length wild typeglucocerebrosidase and variants, homologues, fragments and derivativesthereof which comprise glucocerebrosidase activity.

Any suitable assay as known in the art may be used to assay forglucocerebrosidase activity. Examples of such assays are described inHolleran, W. M., Takagi, Y., Imokawa, G., Jackson, S., Lee, J. M.,Elias, P. M. 1992. β-Glucocerebrosidase activity in murine epidermis:characterization and localization in relation to differentiation. J.Lipid Res. 33:1201-1209 and Holleran, W. M., Takagi, Y., Menon, G. K.,Jackson, S. M., Lee, J. M., Feingold, K. R., Elias, P. M. 1994.Permeability barrier requirements regulate epidermalβ-glucocerebrosidase. J. Lipid Res. 35:905-912.

In brief, an assay for glucocerebrosidase activity may comprise use ofan assay buffer consisting of citrate-phosphate buffer (pH 5.6, and0.54% sodium taurocholate, unless otherwise indicated). The enzymesolution is preheated to 37° C. in the assay buffer (50 n1); reactionsare initiated by addition of 50 μl substrate solution (0.5 mM 4-MUG inassay buffer); incubated for 60 min (37° C.); and terminated with 1.25ml 200 mM carbonate-bicarbonate buffer, pH 10.5. Enzyme activity isdetermined at various pHs (pH 3.2-7.0) as the production of fluorescent4-MU from the β-D-glucoside substrate (4-MUG). Fluorescence is measured(Ex=360 nm, Em=450 nm) with a Perkin-Elmer spectrofluorimeter. Astandard 4-MU solution (0-300 nM) in carbonate-bicarbonate buffer wasused for calibration.

An assay as described in detail at Example 12 below may also be employedto assay glucocerebrosidase activity.

Homologues

The polypeptides disclosed include homologous sequences obtained fromany source, for example related viral/bacterial proteins, cellularhomologues and synthetic peptides, as well as variants or derivativesthereof.

In the context of the present document, a homologous sequence orhomologue is taken to include an amino acid sequence which is at least60, 70, 80 or 90% identical, such as at least 95 or 98% identical at theamino acid level over at least 30, such as 50, 70, 90 or 100 amino acidswith GnT I or polypeptide of interest such as glucocerebrosidase, as thecase may be, for example as shown in the sequence listing herein. In thecontext of this document, a homologous sequence is taken to include anamino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80or 90% identical, such as at least 95 or 98% identical at the amino acidlevel, such as over at least 15, 25, 35, 50 or 100, such as 200, 300,400 or 500 amino acids with the sequence of GnT I or polypeptide ofinterest such as glucocerebrosidase.

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present document it is possible to express homologyin terms of sequence identity. In some embodiments, the sequenceidentity is determined relative to the entirety of the length therelevant sequence, i.e., over the entire length or full length sequenceof the relevant gene, for example.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,the default values may be used when using such software for sequencecomparisons. For example when using the GCG Wisconsin Bestfit package(see below) the default gap penalty for amino acid sequences is −12 fora gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410 ) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). The public default values for theGCG package, or in the case of other software, the default matrix, suchas BLOSUM62 may be used.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, such as % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Variants and Derivatives

The terms “variant” or “derivative” in relation to the amino acidsequences as described here includes any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acids from or to the sequence. For example, the resultantamino acid sequence retains substantially the same activity as theunmodified sequence, such as having at least the same activity as themutant GnT I polypeptide or polypeptide of interest such asglucocerebrosidase protein shown in the sequence listings.

Polypeptides having the amino acid sequence shown in the Examples, orfragments or homologues thereof may be modified for use in the methodsand compositions described here. Typically, modifications are made thatmaintain the biological activity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains the biologicalactivity of the unmodified sequence. Amino acid substitutions mayinclude the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

Natural variants of mutant GnT I and polypeptide of interest such asglucocerebrosidase are likely to comprise conservative amino acidsubstitutions. Conservative substitutions may be defined, for exampleaccording to the Table below Amino acids in the same block in the secondcolumn and in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W YFragments

Polypeptides disclosed here and useful as markers also include fragmentsof the above mentioned full length polypeptides and variants thereof,including fragments of the sequences set out in the sequence listings.

Polypeptides also include fragments of the full length sequence of themutant GnT I polypeptide GnT I polypeptide, as the case may be. Suchfragments may comprise at least one epitope. Methods of identifyingepitopes are well known in the art. Fragments will typically comprise atleast 6 amino acids, such as at least 10, 20, 30, 50 or 100 amino acids.

Included are fragments comprising, such as consisting of, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150, or moreresidues from a mutant GnT I amino acid sequence or polypeptide ofinterest such as glucocerebrosidase amino acid sequence.

Polypeptide fragments of the mutant GnT I proteins and polypeptide ofinterest such as glucocerebrosidase proteins and allelic and speciesvariants thereof may contain one or more (e.g. 5, 10, 15, or 20)substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions occur,for example in different species, such as less than 50%, 40% or 20% ofthe amino acid residues depicted in the sequence listings are altered.

Mutant GnT I and/or polypeptide of interest such as glucocerebrosidase,and fragments, homologues, variants and derivatives, may be made byrecombinant means. However, they may also be made by synthetic meansusing techniques well known to skilled persons such as solid phasesynthesis. The proteins may also be produced as fusion proteins, forexample to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6xHis, GAL4(DNA binding and/or transcriptional activation domains) andβ-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences. Thefusion protein may be such that it does not hinder the function of theprotein of interest sequence. Proteins may also be obtained bypurification of cell extracts from animal cells.

The polypeptide of interest such as glucocerebrosidase or mutant GnT Ipolypeptide, variants, homologues, fragments and derivatives disclosedhere may be in a substantially isolated form. It will be understood thatsuch polypeptides may be mixed with carriers or diluents which will notinterfere with the intended purpose of the protein and still be regardedas substantially isolated. A polypeptide of interest such asglucocerebrosidase or mutant GnT I variant, homologue, fragment orderivative may also be in a substantially purified form, in which caseit will generally comprise the protein in a preparation in which morethan 90%, e.g. 95%, 98% or 99% of the protein in the preparation is aprotein.

The polypeptide of interest such as glucocerebrosidase or mutant GnT Ipolypeptides variants, homologues, fragments and derivatives disclosedhere may be labelled with a revealing label. The revealing label may beany suitable label which allows the polypeptide, etc to be detected.Suitable labels include radioisotopes, e.g. ¹²⁵I, enzymes, antibodies,polynucleotides and linkers such as biotin. Labelled polypeptides may beused in diagnostic procedures such as immunoassays to determine theamount of a polypeptide in a sample. Polypeptides or labelledpolypeptides may also be used in serological or cell-mediated immuneassays for the detection of immune reactivity to said polypeptides inanimals and humans using standard protocols.

Mutant GnT I or polypeptide of interest such as glucocerebrosidasepolypeptide, variants, homologues, fragments and derivatives disclosedhere, optionally labelled, my also be fixed to a solid phase, forexample the surface of an immunoassay well or dipstick. Such labelledand/or immobilised polypeptides may be packaged into kits in a suitablecontainer along with suitable reagents, controls, instructions and thelike. Such polypeptides and kits may be used in methods of detection ofantibodies to the polypeptides or their allelic or species variants byimmunoassay.

Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

The polypeptide of interest such as glucocerebrosidase or mutant GnT Ipolypeptides variants, homologues, fragments and derivatives disclosedhere may be used in in vitro or in vivo cell culture systems to studythe role of their corresponding genes and homologues thereof in cellfunction, including their function in disease. For example, truncated ormodified polypeptides may be introduced into a cell to disrupt thenormal functions which occur in the cell. The polypeptides may beintroduced into the cell by in situ expression of the polypeptide from arecombinant expression vector (see below). The expression vectoroptionally carries an inducible promoter to control the expression ofthe polypeptide.

The use of appropriate host cells, such as insect cells or mammaliancells, is expected to provide for such post-translational modifications(e.g. myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products. Such cellculture systems in which the polypeptide of interest such asglucocerebrosidase or mutant GnT I polypeptide, variants, homologues,fragments and derivatives disclosed here are expressed may be used inassay systems to identify candidate substances which interfere with orenhance the functions of the polypeptides in the cell.

Mutant Gnt I Nucleic Acids

The CHO cells and cell lines comprise mutant GnT I nucleic acids.

We therefore provide generally for a mutant GnT I nucleic acid, togetherwith fragments, homologues, variants and derivatives thereof. Thesenucleic acid sequences may encode the polypeptide sequences disclosedhere, and particularly in the sequence listings.

The polynucleotide may comprise a mutant GnT I nucleic acid. The mutantGnT I nucleic acid may comprise one or more point mutations in the wildtype GnT I sequence. Such mutations may result in corresponding changesto the amino acid sequence, or introduce stop codons and prematuretermination of translation of the GnT I mRNA.

The mutant GnT I nucleic acid may comprise a mutation resulting in astop codon, which results in a mutant GnT I polypeptide being shorterthan a wild type GnT I polypeptide. The length of the mutant GnT Ipolypeptide may be 90% or less, 80% or less, 70% or less, etc than thewild type sequence.

For example, a mutant GnT I nucleic acid may encode a mutant GnT Ipolypeptide that is missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100 or more C-terminal residues.

The mutant GnT I nucleic acid may for example comprise a sequence setout in SEQ ID NO: 1. This is the mutant GnT I nucleic acid sequence fromthe cell line JW152. The mutant GnT I nucleic acid may comprise a GnT Isequence comprising a mutation set out at column 2 of Table E1 below.

In particular, we provide for nucleic acids or polynucleotides whichencode any of the GnT I polypeptides disclosed here. Thus, the term “GnTI sequence” should be construed accordingly. However, such a nucleicacid or polynucleotide may comprise a sequence set out as SEQ ID NO: 1,or a sequence encoding a of the corresponding polypeptide, and afragment, homologue, variant or derivative of such a nucleic acid. Theabove terms therefore may be taken to refer to these sequences.

Homologues, Variants, Derivatives, and Fragments

We also provide for nucleic acids or polynucleotides which encode any ofthe polypeptides of interest such as glucocerebrosidase polypeptidesdisclosed here. Thus, the term “glucocerebrosidase sequence” should beconstrued accordingly. However, such a nucleic acid or polynucleotidemay comprise a sequence set out as GenBank Accession NumbersNP_(—)000148.2 or NM_(—)000157 as the case may be, or a sequenceencoding a of the corresponding polypeptide, and a fragment, homologue,variant or derivative of such a nucleic acid. The above terms thereforemay be taken to refer to these sequences.

Polynucleotide

As used here in this document, the terms “polynucleotide”, “nucleotide”,and nucleic acid are intended to be synonymous with each other.“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

Variants, Derivatives and Homologues

The polypeptide of interest such as glucocerebrosidase and mutant GnT Ipolynucleotides described here may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present document, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span ofpolynucleotides.

Where the polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the methodsand compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleotides from or to the sequence.

As indicated above, with respect to sequence identity, a “homologue” hasfor example at least 5% identity, at least 10% identity, at least 15%identity, at least 20% identity, at least 25% identity, at least 30%identity, at least 35% identity, at least 40% identity, at least 45%identity, at least 50% identity, at least 55% identity, at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity, or at least 95% identity to the relevant sequence shown in thesequence listings.

There may be at least 95% identity, such as at least 96% identity, suchas at least 97% identity, such as at least 98% identity, such as atleast 99% identity. Nucleotide homology comparisons may be conducted asdescribed above. A sequence comparison program that may be used is theGCG Wisconsin Bestfit program described above. The default scoringmatrix has a match value of 10 for each identical nucleotide and −9 foreach mismatch. The default gap creation penalty is −50 and the defaultgap extension penalty is −3 for each nucleotide.

In some embodiments, a mutant GnT I polynucleotide has at least 90% ormore sequence identity to a sequence shown as SEQ ID NO: 1. The mutantGnT I polynucleotide may have 60% or more, such as 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more or 98% or more sequence identity to a sequence shown as SEQID NO: 1.

In other embodiments, a glucocerebrosidase polynucleotide has at least90% or more sequence identity to a sequence having GenBank AccessionNumber NM_(—)000157. The mutant GnT I polynucleotide may have 60% ormore, such as 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more or 98% or more sequenceidentity to a sequence having GenBank Accession Number NM_(—)000157.

Hybridisation

We further describe polypeptide of interest such as glucocerebrosidaseand mutant GnT I nucleotide sequences that are capable of hybridisingselectively to any of the sequences presented herein, or any variant,fragment or derivative thereof, or to the complement of any of theabove. Nucleotide sequences are such as at least 15 nucleotides inlength, such as at least 20, 30, 40 or 50 nucleotides in length.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotidesequences presented herein, or to their complement, will be generally atleast 70%, such as at least 80 or 90% or such as at least 95% or 98%homologous to the corresponding nucleotide sequences presented hereinover a region of at least 20, such as at least 25 or 30, for instance atleast 40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridisable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide isfound to hybridize to the probe at a level significantly abovebackground. The background hybridization may occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screened. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10 fold, such as less than 100 fold asintense as the specific interaction observed with the target DNA. Theintensity of interaction may be measured, for example, by radiolabellingthe probe, e.g. with ³²P.

Hybridisation conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridisation can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridisation can be used to identifyor detect similar or related polynucleotide sequences.

In a one aspect, we disclose nucleotide sequences that can hybridise toa polypeptide of interest such as glucocerebrosidase and or mutant GnT Inucleic acid, or a fragment, homologue, variant or derivative thereof,under stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}).

Where a polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the presentdisclosure. Where the polynucleotide is single-stranded, it is to beunderstood that the complementary sequence of that polynucleotide isalso disclosed and encompassed.

Polynucleotides which are not 100% homologous to the sequences disclosedhere but fall within the disclosure can be obtained in a number of ways.Other variants of the sequences described herein may be obtained forexample by probing DNA libraries made from a range of individuals, forexample individuals from different populations. In addition, otherviral/bacterial, or cellular homologues particularly cellular homologuesfound in mammalian cells (e.g. rat, mouse, bovine and primate cells),may be obtained and such homologues and fragments thereof in generalwill be capable of selectively hybridising to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of SEQ IDNO: 1 under conditions of medium to high stringency. Similarconsiderations apply to obtaining species homologues and allelicvariants of polypeptide of interest such as glucocerebrosidase andmutant GnT I.

The polynucleotides described here may be used to produce a primer, e.g.a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, such as at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides as used herein. Fragments may be less than 500, 200,100, 50 or 20 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the sequence which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom an animal or human cell, performing a polymerase chain reactionunder conditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

Gaucher's Disease

[The text in this section is adapted from Gaucher's disease. (2010,March 23). In Wikipedia, The Free Encyclopedia. Retrieved 13:15, Mar.25, 2010, from en.wikipedia.org/w/index.php?title=Gaucher%27s_disease&oldid=351623575]

Gaucher's disease is a genetic disease in which a fatty substance(lipid) accumulates in cells and certain organs. Gaucher's disease isthe most common of the lysosomal storage diseases.[1]:536 It is causedby a hereditary deficiency of the enzyme glucocerebrosidase (also knownas acid β-glucosidase). The enzyme acts on a fatty substanceglucocerebroside (also known as glucosylceramide). When the enzyme isdefective, the substance accumulates, particularly in cells of themononuclear cell lineage. Fatty material can collect in the spleen,liver, kidneys, lungs, brain and bone marrow.

Symptoms may include enlarged spleen and liver, liver malfunction,skeletal disorders and bone lesions that may be painful, severeneurologic complications, swelling of lymph nodes and (occasionally)adjacent joints, distended abdomen, a brownish tint to the skin, anemia,low blood platelets and yellow fatty deposits on the white of the eye(sclera). Persons affected most seriously may also be more susceptibleto infection. Some forms of Gaucher's disease may be treated with enzymereplacement therapy.

The disease is caused by a recessive mutation in a gene located onchromosome 1 and affects both males and females. About 1 in 100 peoplein the United States are carriers of the most common type of Gaucherdisease, while the carrier rate among Ashkenazi Jews is 1 in 154.[2]

The disease is named after the French doctor Philippe Gaucher, whooriginally described it in 1882.

Classification

Gaucher's disease has three common clinical subtypes.

Type I (or non-neuropathic type) is the most common form of the disease,occurring in approximately 1 in 50,000 live births. It occurs most oftenamong persons of Ashkenazi Jewish heritage. Symptoms may begin early inlife or in adulthood and include enlarged liver and grossly enlargedspleen (together hepatosplenomegaly); the spleen can rupture and causeadditional complications. Skeletal weakness and bone disease may beextensive. Spleen enlargement and bone marrow replacement cause anemia,thrombocytopenia and leukopenia. The brain is not affected, but theremay be lung and, rarely, kidney impairment. Patients in this groupusually bruise easily (due to low levels of platelets) and experiencefatigue due to low numbers of red blood cells. Depending on diseaseonset and severity, type 1 patients may live well into adulthood. Manypatients have a mild form of the disease or may not show any symptoms.

Type II (or acute infantile neuropathic Gaucher's disease) typicallybegins within 6 months of birth and has an incidence rate ofapproximately 1 in 100,000 live births. Symptoms include an enlargedliver and spleen, extensive and progressive brain damage, eye movementdisorders, spasticity, seizures, limb rigidity, and a poor ability tosuck and swallow. Affected children usually die by age 2.

Type III (the chronic neuropathic form) can begin at any time inchildhood or even in adulthood, and occurs in approximately 1 in 100,000live births. It is characterized by slowly progressive but milderneurologic symptoms compared to the acute or type 2 version. Majorsymptoms include an enlarged spleen and/or liver, seizures, poorcoordination, skeletal irregularities, eye movement disorders, blooddisorders including anemia and respiratory problems. Patients often liveinto their early teen years and adulthood.

These subtypes have come under some criticism for not taking account ofthe full spectrum of observable symptoms (the phenotypes.[1]) There arealso compound heterozygous variations which considerably increase thecomplexity of predicting disease course.

Signs and Symptoms

Painless hepatomegaly and splenomegaly; the size of the spleen can be1500-3000 ml, as opposed to the normal size of 50-200 ml.

Hypersplenism: the rapid and premature destruction of blood cells,leading to anemia, neutropenia and thrombocytopenia (with an increasedrisk of infection and bleeding)

Cirrhosis of the liver is rare

Neurological symptoms occur only in some types of Gaucher's (see below):

Type II: serious convulsions, hypertonia, mental retardation, apnea.

Type III: muscle twitches known as myoclonus, convulsions, dementia,ocular muscle apraxia.

Osteoporosis: 75% develop visible bony abnormalities due to theaccumulated glucosylceramide. A deformity of the distal femur in theshape of an Erlenmeyer flask is commonly described (aseptic necrosis ofthe femur joint).

Yellowish-brown skin pigmentation

Pathophysiology

Acid Beta-Glucosidase

The disease is caused by a defect in the housekeeping gene lysosomalglucocerebrosidase (also known as beta-glucosidase, EC 3.2.1.45, PDB1OGS) on the first chromosome (1q21). The enzyme is a 55.6 KD, 497 aminoacids long protein that catalyses the breakdown of glucocerebroside, acell membrane constituent of red and white blood cells. The macrophagesthat clear these cells are unable to eliminate the waste product, whichaccumulates in fibrils, and turn into Gaucher cells, which appear onlight microscopy to resemble crumpled-up paper.

In the brain (type II and III), glucocerebroside accumulates due to theturnover of complex lipids during brain development and the formation ofthe myelin sheath of nerves.

Different mutations in the beta-glucosidase determine the remainingactivity of the enzyme, and, to a large extent, the phenotype.

Heterozygotes for particular acid beta-glucosidase mutations carry abouta fivefold risk of developing Parkinson's disease, making this the mostcommon known genetic risk-factor for Parkinson's.[2][4] A study of 1525Gaucher patients in the United States suggested that while cancer riskis not elevated, particular malignancies (non-Hodgkin lymphoma, melanomaand pancreatic cancer) occurred at a 2-3 times higher rate. [5]

Genetics

The three types of Gaucher's disease are inherited in an autosomalrecessive fashion. Both parents must be carriers in order for a child tobe affected. If both parents are carriers, there is a one in four, or25%, chance with each pregnancy for an affected child. Geneticcounseling and genetic testing is recommended for families who may becarriers of mutations.

Each type has been linked to particular mutations. In all, there areabout 80 known mutations, grouped into three main types:[6]

Type I (N370S homozygote), the most common, also called the“non-neuropathic” type occurs mainly in Ashkenazi Jews, at 100 times theoccurrence in the general populace. The median age at diagnosis is 28years of age,[7] and life expectancy is mildly decreased[8]. There areno neurological symptoms.

Type II (1 or 2 alleles L444P) is characterized by neurological problemsin small children. The enzyme is hardly released into the lysosomes.Prognosis is dismal: most die before reaching the third birthday.

Type III (also 1-2 copies of L444P, possibly delayed by protectivepolymorphisms) occurs in Swedish patients from the Norrbotten region.This group develops the disease somewhat later, but most die beforetheir 30th birthday.

Diaz et al. suggest that the Gaucher-causing mutations entered theAshkenazi Jewish gene pool in the early Middle Ages (48-55 generationsago).[9]

Diagnosis

A definitive diagnosis is made with genetic testing. As there arenumerous different mutations, sequencing of the beta-glucosidase gene issometimes necessary to confirm the diagnosis. Prenatal diagnosis isavailable, and is useful when there is a known genetic risk factor.

A diagnosis can also be implied by biochemical abnormalities such ashigh alkaline phosphatase, angiotensin-converting enzyme (ACE) andimmunoglobulin levels, or by cell analysis showing “crinkled paper”cytoplasm and glycolipid-laden macrophages.

Some lysosomal enzymes are elevated, including tartrate-resistant acidphosphatase, hexosaminidase, and a human chitinase, chitotriosidase.This latter enzyme has proved to be very useful for monitoring Gaucher'sdisease activity in response to treatment, and may reflect the severityof the disease

Treatment

For type 1 and most type 3 patients, enzyme replacement treatment withintravenous recombinant glucocerebrosidase (imiglucerase) candramatically decrease liver and spleen size, reduce skeletalabnormalities, and reverse other manifestations. This treatment costsapproximately $200,000 annually for a single patient and should becontinued for life or until one's finances are exhausted, whichevercomes sooner. The rarity of the disease means that dose-finding studieshave been difficult to conduct, so there remains controversy over theoptimal dose and dosing frequency. [7] Due to the low incidence, thishas become an orphan drug in many countries, meaning that a governmentrecognizes and accommodates the financial constraints that limitresearch into drugs that address a small population. Velaglucerase alfawas approved by the FDA as an alternative treatment on Feb. 26, 2010.[10]

Successful bone marrow transplantation cures the non-neurologicalmanifestations of the disease, because it introduces a monocytepopulation with active beta-glucosidase. However, this procedure carriessignificant risk and is rarely performed in Gaucher patients. Surgery toremove the spleen (splenectomy) may be required on rare occasions if thepatient is anemic or when the enlarged organ affects the patient'scomfort. Blood transfusion may benefit some anemic patients. Otherpatients may require joint replacement surgery to improve mobility andquality of life. Other treatment options include antibiotics forinfections, antiepileptics for seizures, bisphosphonates for bonelesions, and liver transplants. Substrate reduction therapy may prove tobe effective in stopping Type 2, as it can cross through the bloodbarrier into the brain. There is currently no effective treatment forthe severe brain damage that may occur in patients with types 2 and 3Gaucher disease. Gene therapy may be a future step.

Gaucher's disease has recently become a target for more than one effortat pharmacological chaperoning, which involves the use of orallyadministered drugs that operate at a molecular level. Miglustat is oneof these oral drugs. It was approved for the treatment of this diseasein 2003. As of June 2009, another oral drug, isofagomine tartrate, isunder development.

Epidemiology

The National Gaucher Foundation states that around 1 in 100 people inthe general U.S. population is a carrier for type 1 Gaucher's disease,giving a prevalence of 1 in 40,000: among Ashkenazi Jews the rate ofcarriers is considerably higher, at roughly 1 in 15.[11]

Type 2 Gaucher's disease shows no particular preference for any ethnicgroup. Type 3 Gaucher's disease is especially common in the populationof the Northern Swedish region of Norrbotten where the incidence of thedisease is 1 in 50,000.

History

The disease was first recognised by the French doctor Philippe Gaucher,who originally described it in 1882 and lent his name to thecondition.[3] The biochemical basis for the disease would be elucidatedin 1965.[12] The first effective treatment for the disease, the drugCeredase, was approved by the FDA in June 1995. An improved drug,Cerezyme, was approved by the FDA in 2001 and has replaced the use ofCeredase.

Pharmaceutical Compositions

As disclosed herein, mannose-terminated recombinant proteins such asglucocerebrosidase may be used to treat or prevent disease andinfection, including for example Gaucher's Disease. Othermannose-terminated recombinant proteins may be used to treat or preventtheir cognate diseases, e.g., tumour-specific or tumour-associatedantigens for treatment or prevention of cancer, viral antigens fortreatment or prevention of viral diseases, etc

Mannose-terminated recombinant proteins of interest such asglucocerebrosidase can be administered in a variety of ways includingenteral, parenteral and topical routes of administration. For example,suitable modes of administration include oral, subcutaneous,transdermal, transmucosal, iontophoretic, intravenous, intramuscular,intraperitoneal, intranasal, subdural, rectal, and the like.

In accordance with other embodiments, there is provided a compositioncomprising a mannose-terminated recombinant proteins of interest such asglucocerebrosidase, together with a pharmaceutically acceptable carrieror excipient for the treatment or prevention of disease such asGaucher's Disease, cancer, viral disease, etc.

Suitable pharmaceutically acceptable excipients include processingagents and drug delivery modifiers and enhancers, such as, for example,calcium phosphate, magnesium stearate, talc, monosaccharides,disaccharides, starch, gelatin, cellulose, methyl cellulose, sodiumcarboxymethyl cellulose, dextrose, hydroxypropyl-p-cyclodextrin,polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and thelike, as well as combinations of any two or more thereof. Other suitablepharmaceutically acceptable excipients are described in “Remington'sPharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), incorporatedherein by reference.

Pharmaceutical compositions containing mannose-terminated recombinantproteins of interest such as glucocerebrosidase may be in any formsuitable for the intended method of administration, including, forexample, a solution, a suspension, or an emulsion. Liquid carriers aretypically used in preparing solutions, suspensions, and emulsions.Liquid carriers contemplated for use in the practice include, forexample, water, saline, pharmaceutically acceptable organic solvent (s),pharmaceutically acceptable oils or fats, and the like, as well asmixtures of two or more thereof. The liquid carrier may contain othersuitable pharmaceutically acceptable additives such as solubilizers,emulsifiers, nutrients, buffers, preservatives, suspending agents,thickening agents, viscosity regulators, stabilizers, and the like.Suitable organic solvents include, for example, monohydric alcohols,such as ethanol, and polyhydric alcohols, such as glycols.

Suitable oils include, for example, soybean oil, coconut oil, olive oil,safflower oil, cottonseed oil, and the like. For parenteraladministration, the carrier can also be an oily ester such as ethyloleate, isopropyl myristate, and the like. Compositions may also be inthe form of microparticles, microcapsules, liposomal encapsulates, andthe like, as well as combinations of any two or more thereof.

The mannose-terminated recombinant protein of interest such asglucocerebrosidase may be administered orally, parenterally,sublingually, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. Topicaladministration may also involve the use of transdermal administrationsuch as transdermal patches or ionophoresis devices. The term parenteralas used herein includes subcutaneous injections, intravenous,intramuscular, intrastemal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-propanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e. g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

In accordance with yet other embodiments, we provide methods forinhibiting any activity of envelope glycoprotein (E) domain III, in ahuman or animal subject, the method comprising administering to asubject an amount of a mannose-terminated recombinant proteins ofinterest such as glucocerebrosidase (or composition comprising suchcompound) effective to inhibit the relevant activity in the subject.Other embodiments provide methods for treating disease such as Gaucher'sDisease, in a human or animal subject, comprising administering to thecell or to the human or animal subject an amount of a compound orcomposition as described here effective to inhibit a envelopeglycoprotein (E) domain III activity in the cell or subject. The subjectmay be a human or non-human animal subject. Inhibition of proteinactivity includes detectable suppression of the relevant proteinactivity either as compared to a control or as compared to expectedprotein activity.

Effective amounts of the mannose-terminated recombinant protein ofinterest such as glucocerebrosidase generally include any amountsufficient to detectably inhibit the relevant protein activity by any ofthe assays described herein, by other assays known to those havingordinary skill in the art or by detecting an alleviation of symptoms ina subject afflicted with a disease such as Gaucher's Disease.

Successful treatment of a subject in accordance may result in theinducement of a reduction or alleviation of symptoms in a subjectafflicted with a medical or biological disorder to, for example, haltthe further progression of the disorder, or the prevention of thedisorder. Thus, for example, treatment of disease such as Gaucher'sDisease can result in a reduction in symptoms such as hepatomegaly,splenomegaly, hypersplenism, anemia, neutropenia, thrombocytopenia, riskof infection and bleeding, cirrhosis of the liver is rare, neurologicalsymptoms, Type II symptoms such as serious convulsions, hypertonia,mental retardation, apnea, Type III symptoms such as muscle twitchesknown as myoclonus, convulsions, dementia, ocular muscle apraxia,osteoporosis, deformity of the distal femur, aseptic necrosis of thefemur joint and yellowish-brown skin pigmentation.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. The therapeutically effective amount for a givensituation can be readily determined by routine experimentation and iswithin the skill and judgment of the ordinary clinician.

A therapeutically effective dose will generally be from about 10μg/kg/day to 100 mg/kg/day, for example from about 25 μg/kg/day to about20 mg/kg/day or from about 50 μg/kg/day to about 2 mg/kg/day of amannose-terminated recombinant protein of interest such asglucocerebrosidase, which may be administered in one or multiple doses.

The mannose-terminated recombinant protein of interest such asglucocerebrosidase can also be administered in the form of liposomes. Asis known in the art, liposomes are generally derived from phospholipidsor other lipid substances. Liposomes are formed by mono- ormultilamellar hydrated liquid crystals that are dispersed in an aqueousmedium. Any non-toxic, physiologically acceptable and metabolizablelipid capable of forming liposomes can be used. The present compositionsin liposome form can contain, in addition to a compound, stabilizers,preservatives, excipients, and the like. Lipids which may be usedinclude the phospholipids and phosphatidyl cholines (lecithins), bothnatural and synthetic. Methods to form liposomes are known in the art.See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV,Academic Press, New York, N. W., p. 33 et seq (1976).

While the mannose-terminated recombinant protein of interest such asglucocerebrosidase can be administered as the sole active pharmaceuticalagent, they can also be used in combination with one or more otheragents used in the treatment of disorders. Representative agents usefulin combination with the mannose-terminated recombinant protein ofinterest such as glucocerebrosidase for the treatment of disease such asGaucher's Disease include, for example, recombinant glucocerebrosidase(imiglucerase), Cerazyme and Ceredase as well as any of the othertreatments listed above in “Gaucher's Disease”. Furthermore, any of theprocedures such as surgical procedures listed there under “Treatment”may also be used in combination with administration ofmannose-terminated recombinant protein such as glucocerebrosidase.

When additional active agents are used in combination with themannose-terminated recombinant protein of interest such asglucocerebrosidase, the additional active agents may generally beemployed in therapeutic amounts as indicated in the PHYSICIANS'DESKREFERENCE (PDR) 53rd Edition (1999), which is incorporated herein byreference, or such therapeutically useful amounts as would be known toone of ordinary skill in the art.

The mannose-terminated recombinant protein of interest such asglucocerebrosidase and the other therapeutically active agents can beadministered at the recommended maximum clinical dosage or at lowerdoses. Dosage levels of the active mannose-terminated recombinantprotein of interest such as glucocerebrosidase in the compositions maybe varied so as to obtain a desired therapeutic response depending onthe route of administration, severity of the disease and the response ofthe patient. The combination can be administered as separatecompositions or as a single dosage form containing both agents. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are given at the same time or differenttimes, or the therapeutic agents can be given as a single composition.

Bioavailability

The compounds disclosed here (and combinations) are in some embodimentsorally bioavailable. Oral bioavailablity refers to the proportion of anorally administered drug that reaches the systemic circulation. Thefactors that determine oral bioavailability of a drug are dissolution,membrane permeability and metabolic stability. Typically, a screeningcascade of firstly in vitro and then in vivo techniques is used todetermine oral bioavailablity.

Dissolution, the solubilisation of the drug by the aqueous contents ofthe gastro-intestinal tract (GIT), can be predicted from in vitrosolubility experiments conducted at appropriate pH to mimic the GIT. Themannose-terminated recombinant protein of interest such asglucocerebrosidase may in some embodiments have a minimum solubility of50 mg/ml. Solubility can be determined by standard procedures known inthe art such as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.

Membrane permeability refers to the passage of the compound through thecells of the GIT. Lipophilicity is a key property in predicting this andis defined by in vitro Log D_(7.4) measurements using organic solventsand buffer. The mannose-terminated recombinant protein of interest suchas glucocerebrosidase may have a Log D_(7.4) of −2 to +4 or −1 to +2.The log D can be determined by standard procedures known in the art suchas described in J. Pharm. Pharmacol. 1990, 42:144.

Cell monolayer assays such as CaCO₂ add substantially to prediction offavourable membrane permeability in the presence of efflux transporterssuch as p-glycoprotein, so-called caco-2 flux. The mannose-terminatedrecombinant protein of interest such as glucocerebrosidase may have acaco-2 flux of greater than 2×10⁻⁶cms⁻¹, for example greater than5×10⁻⁶cms⁻¹. The caco flux value can be determined by standardprocedures known in the art such as described in J. Pharm. Sci, 1990,79, 595-600.

Metabolic stability addresses the ability of the GIT or the liver tometabolise compounds during the absorption process: the first passeffect. Assay systems such as microsomes, hepatocytes etc are predictiveof metabolic liability. The compounds of the Examples may in someembodiments show metabolic stability in the assay system that iscommensurate with an hepatic extraction of less than 0.5. Examples ofassay systems and data manipulation are described in Curr. Opin. DrugDisc. Devel., 201, 4, 36-44, Drug Met. Disp., 2000, 28, 1518-1523.

Because of the interplay of the above processes further support that adrug will be orally bioavailable in humans can be gained by in vivoexperiments in animals. Absolute bioavailability is determined in thesestudies by administering the compound separately or in mixtures by theoral route. For absolute determinations (% absorbed) the intravenousroute is also employed. Examples of the assessment of oralbioavailability in animals can be found in Drug Met. Disp., 2001, 29,82-87; J. Med Chem, 1997, 40, 827-829, Drug Met. Disp., 1999, 27,221-226.

The term “pharmaceutically acceptable carrier” as used herein generallyrefers to organic or inorganic materials, which cannot react with activeingredients. The carriers include but are not limited to sugars, such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodiumcarboxymethycellulose, ethylcellulose and cellulose acetates; powderedtragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;calcium sulfate; vegetable oils, such as peanut oil, cotton seed oil,sesame oil, olive oil, corn oil and oil of theobroma; polyols such aspropylene glycol, glycerine, sorbitol, mannitol, and polyethyleneglycol; agar; alginic acids; pyrogen-free water; isotonic saline; andphosphate buffer solution; skim milk powder; as well as other non-toxiccompatible substances used in pharmaceutical formulations. Wettingagents and lubricants such as sodium lauryl sulfate, as well as coloringagents, flavoring agents, lubricants, excipients, tabletting agents,stabilizers, anti-oxidants and preservatives, can also be present.

The term “therapeutically effective amount” as used herein generallyrefers to an amount of an agent, for example the amount of a compound asan active ingredient, that is sufficient to effect treatment as definedherein when administered to a subject in need of such treatment. Atherapeutically effective amount of a compound, salt, derivative, isomeror enantiomer of the present invention will depend upon a number offactors including, for example, the age and weight of the subject, theprecise condition requiring treatment and its severity, the nature ofthe formulation, and the route of administration, and will ultimately beat the discretion of the attendant physician or veterinarian.

However, an effective amount mannose-terminated recombinant protein forthe treatment of disorders such as Gaucher's Disease, may generally bein the range of about 10 to about 40 mg/kg body weight of recipient(mammal) per day and more usually about 40 mg/kg body weight per day.Thus, for a 70 kg adult subject, the actual amount per day wouldtypically be about 2,800 mg, and this amount may be given in a singledose per day or more usually in a number (such as two, three, four, fiveor six) of sub-doses per day such that the total daily dose is the same.An effective amount of a salt of the present invention may be determinedas a proportion of the effective amount of the compound per se.

The term “treatment” as used herein refers to any treatment of acondition or disease in an animal, particularly a mammal, moreparticularly a human, and includes: preventing the disease or conditionfrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it; inhibiting the disease orcondition, i.e. arresting its development; relieving the disease orcondition, i.e. causing regression of the condition; or relieving theconditions caused by the disease, i.e. symptoms of the disease.

Chemical Derivative

The term “derivative” or “derivatised” as used herein includes chemicalmodification of a compound. Illustrative of such chemical modificationswould be replacement of hydrogen by a halo group, an alkyl group, anacyl group or an amino group.

Chemical Modification

In one embodiment, the compound may be a chemically modified compound.

The chemical modification of a compound may either enhance or reducehydrogen bonding interaction, charge interaction, hydrophobicinteraction, Van Der Waals interaction or dipole interaction between thecompound and the target.

In one aspect, the identified compound may act as a model (for example,a template) for the development of other compounds.

Individual

The compounds are delivered to individuals. As used herein, the term“individual” refers to vertebrates, particularly members of themammalian species. The term includes but is not limited to domesticanimals, sports animals, primates and humans.

Further Aspects

Further aspects and embodiments of the invention are now set out in thefollowing numbered Paragraphs; it is to be understood that the inventionencompasses these aspects:

Paragraph 1. A method of expressing a recombinant protein comprisingmannose-terminated N-glycans from a host cell, the method comprising:(a) providing a host cell comprising a Chinese Hamster Ovary (CHO) cellwhich is selected with Ricinus communis agglutinin I (RCA-I) or adescendent thereof; (b) introducing a nucleic acid encoding arecombinant protein into the host cell; and (c) expressing therecombinant protein from the host cell, in which the expressedrecombinant protein comprises a mannose-terminated glycan structure inwhich the method does not include a step of introducing functional GnT-Iinto the host cell.

Paragraph 2. A method according to Paragraph 1, in which the method doesnot include a step of introducing a nucleic acid encoding functionalGnT-I into the host cell.

Paragraph 3. A method according to Paragraphs 1 or 2, in which the CHOcell selected with RCA-I or descendent thereof comprises a mutation inthe GnT-I gene.

EXAMPLES Example 1 Cell Culture

Chinese hamster ovary-K1 (CHO-K1) cells are originally obtained from Dr.Donald K. MacCallum (University of Michigan Medical Scholl, Ann Arbor,Mich.).

Parental and mutant CHO cells including JW152 cells are cultured inDulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetalbovine serum (FBS) at 37° C. in a humidified incubator with 5% CO₂.

Lec1.3 cells (Lec 1 cells) are kindly provided by Dr. P. Stanley (AlbertEinstein College of Medicine, NY) and cultured in α-MEM (Gibco)supplemented with Proline (40 mg/L) (Invitrogen/Gibco) and 10% FBS.

Example 2 Isolation of RCA-I-Resistant CHO Cells

RCA-I-resistant CHO cells, including JW152, are isolated as describedbelow.

CHO-K1 cells are cultured in 6-well plates to confluence before culturemedia is changed to serum-free DMEM. Ricinus communis agglutinin I(RCA-I, EY Laboratories) is added into the media to reach a finalconcentration of 10 μg/ml and incubated with cells overnight.

Then the serum-free DMEM containing RCA-I is replaced with fresh DMEMwith 10% FBS. Nine days later, colonies of the CHO cells that survivedthe RCA-I treatment are picked and cultured in 24 well plates.

These cells are then subjected to agglutination tests using RCA-I toconfirm the mutant cells no longer react with RCA-I.

Example 3 Cell Line JW152

As described above under “Isolation of RCA-I-Resistant CHO Cells”, cellline JW152 is isolated from CHO-K1 cells by selection using a cytotoxiclectin, RCA-I. JW152 has been adapted in suspension culture and grown inserum-free medium.

The cell line JW152 was deposited on 11 Dec. 2008 at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, United States of America under the accession number PTA-9657as the International Deposition Number under the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

Example 4 Molecular Cloning and Sequencing Analysis of GnT I cDNA inRCA-I-Resistant CHO Cells, Including JW152

For each cell line, including JW152, 1×10⁷ cells are pelleted and rinsedin PBS. Total RNA is extracted from the pellet using the RNAqueous kit(Ambion). cDNA is then synthesized through reverse transcription usingMoloney Murine Leukemia virus (MMLV) reverse transcriptase (Promega)according to the manufacturer's recommendations

The GnT I amplicon from each cell lines' cDNA is obtained throughpolymerase chain reaction (PCR) using PFX (Invitrogen). This is thencloned into pcDNA 3.1 expression vector and sequenced. A minimum of fourclones from each mutant line are sequenced.

All plasmid purifications are carried out using mini or midi-preparationkits from Promega. Constructs are sequenced using ABI Prism 3100 GeneticAnalyzer (Applied Biosystems) after cycle sequencing with Big Dye 3.1(Applied Biosystems).

Results are shown in the following Example.

Example 5 Mutations in Gnt-I Gene of Rca-I-Resistant Cho Cells,Including JW152

TABLE E1 Table of mutations found in GnT1 mutants from clones derivedfrom RCA-I screening. Nine CHO glycosylation mutants with differentmutations in the GnT1 gene. Point mutations leading to loss in functionwere found in four cell lines (JW80, JW36, KFC15002, KFC15071). A pointinsertion resulting in the generation of a stop codon was found in KFC5008. Mutations leading to a premature stop codon were also found inanother four cell lines. (KFC5026, KFC20011, KFC15047). PolypeptideMutant CHO DNA Mutation Cell Line Mutation (position, mutation) CommentJW80 G1300C Position 434 Domain 2 Ala →Pro β14 JW36 A638C Position 213Disruption of DxD Asp → Ala Motif, similar to JW98, JW191 KFC15002 C784GPosition 262 Domain 1 β6, similar Arg →Gly to KFC15008 (aka 15008),KFC7501 (aka 7501) and KFC12008 KFC15071 T811A Position 271 Domain 1 Trp→ Arg β7 KFC5008 _706C Insertion at 706 bp Stop codon generated Frameshift from at 245 a. a. 236 aa Asp→STOP KFC5026 G246A Position 82 365a.a. missing from Trp→STOP C terminal KFC20011 G258A Position 86 361a.a. missing from Trp→STOP C terminal KFC 15047 A859T Position 287 160a.a. missing, Lys→STOP similar to KFC15026 and KFC15072 The mutation ofGnT1 of JW152 is determined as described above and shown in thecontinuation of Table E1 below. Table of mutations found in JW152 GnT1mutant derived from RCA-I screening. The mutation in GnT1 of JW152 leadsto a premature stop codon. Mutant DNA Polypeptide Mutation CHO Cell LineMutation (position, mutation) Comment JW152 C1015T Position 339 108 a.a.missing Gln→STOP from C terminal

JW152 GnT1 Mutation

Example 6 JW152 GnT1 Nucleic Acid Sequence

The coding region of N-acetylglucoaminyltransferase I (Mgat1, GenBank:AF343963) mRNA isolated from the CHO JW152 cells is shown below.

In these mutant cells, a C to T point mutation at position 1015 wasidentified (shown in bold) (SEQ ID NO.1):

ATGCTGAAGAAGCAGTCTGCAGGGCTTGTGCTTTGGGGTGCTATCCTCTTTGTGGGCTGGAATGCCCTGCTGCTCCTCTTCTTCTGGACACGCCCAGCCCCTGGCAGGCCCCCCTCAGATAGTGCTATCGATGATGACCCTGCCAGCCTCACCCGTGAGGTGTTCCGCCTGGCTGAGGACGCTGAGGTGGAGTTGGAGCGGCAGCGGGGGCTGTTGCAGCAAATCAGGGAGCATCATGCTTTGTGGAGACAGAGGTGGAAAGTGCCCACCGTGGCCCCTCCAGCCTGGCCCCGTGTGCCTGCGACCCCCTCACCAGCCGTGATCCCCATCCTGGTCATTGCCTGTGACCGCAGCACTGTCCGGCGCTGCTTGGATAAGTTGTTGCACTATCGGCCCTCAGCTGAGCATTTCCCCATCATTGTCAGCCAGGACTGCGGGCACGAAGAGACAGCACAGGTCATTGCTTCCTATGGCAGTGCAGTCACACACATCCGGCAGCCAGACCTGAGTAACATCGCTGTGCCCCCAGACCACCGCAAGTTCCAGGGTTACTACAAGATCGCCAGGCACTACCGCTGGGCACTGGGCCAGATCTTCAACAAGTTCAAGTTCCCAGCAGCTGTGGTAGTGGAGGACGATCTGGAGGTGGCACCAGACTTCTTTGAGTACTTCCAGGCCACCTACCCACTGCTGAGAACAGACCCCTCCCTTTGGTGTGTGTCTGCTTGGAATGACAATGGCAAGGAGCAGATGGTAGACTCAAGCAAACCTGAGCTGCTCTATCGAACAGACTTTTTTCCTGGCCTTGGCTGGCTGCTGATGGCTGAGCTGTGGACAGAGCTGGAGCCCAAGTGGCCCAAGGCCTTCTGGGATGACTGGATGCGCAGACCTGAGCAGCGGAAGGGGCGGGCCTGTATTCGTCCAGAAATTTCAAGAACGATGACCTTTGGCCGTAAGGGTGTGAGCCATGGGCAGTTCTTTGATCAGCATCTTAAGTTCATCAAGCTGAACCAGTAGTTCGTGTCTTTCACCCAGTTGGATTTGTCATACTTGCAGCGGGAGGCTTATGACCGGGATTTCCTTGCCCGTGTCTATAGTGCCCCCCTGCTACAGGTGGAGAAAGTGAGGACCAATGATCAGAAGGAGCTGGGGGAGGTGCGGGTACAGTACACTAGCAGAGACAGCTTCAAGGCCTTTGCTAAGGCCCTGGGTGTCATGGATGACCTCAAGTCTGGTGTCCCCAGAGCTGGCTACCGGGGCGTTGTCACTTTCCAGTTCAGGGGTCGACGTGTCCACCTGGCACCCCCACAAACCTGGGAAGGCTATGATCCTAGCTGGAATTAG

Example 7 JW152 GnT1 Amino Acid Sequence

The N-acetylglucoaminyltransferase I (GnT I) protein encoded by themutated gene in CHO JW152 cells, with sequence shown above, has thesequence shown below.

As a result of the point mutation (C1015T), JW152 cells only produce atruncated version of GnT I which contains only 338 amino acids ratherthan the normal protein that contains 447 amino acids (SEQ ID NO.2). TheC-terminal portion in bold is not translated in JW152 cells.

MLKKQSAGLVLWGAILFVGWNALLLLFFWTRPAPGRPPSDSAIDDDPASLTREVFRLAEDAEVELERQRGLLQQIREHHALWRQRWKVPTVAPPAWPRVPATPSPAVIPILVIACDRSTVRRCLDKLLHYRPSAEHFPIIVSQDCGHEETAQVIASYGSAVTHIRQPDLSNIAVPPDHRKFQGYYKIARHYRWALGQIFNKFKEPAAVVVEDDLEVAPDFFEYFQATYPLLRTDPSLWCVSAWNDNGKEQMVDSSKPELLYRTDFFPGLGWLLMAELWTELEPKWPKAFWDDWMRRPEQRKGRACIRPEISRTMTFGRKGVSHGQFFDQHLKFIKLNQQFVSFTQLDLSYLQREAYDRDFLARVYSAPLLQVEKVRTNDQKELGEVRVQYTSRDSFKAFAKALGVMDDLKSGVPRAGYRGVVT FQFRGRRVHLAPPQTWEGYDPSWN

Example 8 Glucocerebrosidase Sequences

The amino acid sequence for Glucocerebrosidase (GenBank AccessionNumber: NP_(—)000148.2, SEQ ID NO. 3) is:

>gi|54607043|ref|NP_000148.2| glucocerebrosidaseprecursor [Homo sapiens] acid beta-glucosidase geneMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ

The DNA sequence for the coding frame of Glucocerebrosidase (GenBankAccession Number: NM_(—)000157, SEQ ID NO.4) is:

NM 000157. Homo sapiens gluc . . . [gi: 54607042]>gi|54607042: 151-1761 Homo sapiens glucosidase,beta; acid (includes glucosylceramidase) (GBA),transcript variant 1, mRNAATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGTGA

Example 9 Cloning and Attachment of Restriction Sites

The coding sequence is amplified using PCR with the following primers toattach EcoRI and XhoI restriction sites:

Forward primer: (SEQ ID NO. 5) GCGGAATTCGCCACCATGGCTGGCAGCCTCACAGGAReverse primer: (SEQ ID NO. 6) CTCGAGTCACTGGCGACGCCACAG

Example 10 Production of Expression Construct and Transient Expression

The coding frame is cloned into a mammalian expression vector pcDNA3.1(+) (Invitrogen, USA) using the restriction enzymes, EcoRI and XhoI.

The resulting expression construct may be used for transient expressionof recombinant human glucocerebrosidase in parental and mutant CHO celllines using the following procedure.

Unless specified, 1 μg of DNA construct that expressesglucocerebrosidase is transfected into JW-152 mutant cells withLipofectamine (Invitrogen) according to the manufacturer's protocols.

Two days after transfection, conditioned culture media from thetransfected cells are collected. The concentrations of recombinantglucocerebrosidase in each transfection sample are determined bystandard ELISA.

Example 11 Cell Transfection and Selection

The expression plasmid is transfected into the JW152 cells grown insix-well plates using Lipofectamine One day after transfection, thecells are put into selection medium. Selection medium consists of DMEMwith 10% fetal bovine serum and 800 μg/ml of G418 antibiotic.

After 10-15 days, when the untransfected cells under selection mediumhave died, the transfected cells are deemed to be the stable pool andare then trypsinised and seeded sparsely to yield distinct singlecolonies which are picked and grown in 96 well plates.

After making duplicates plates, the supernatant from the 96 well platesis analysed using dot blot assay and an anti-glucocerebrosidase antibody(AbD Serotec, Japan) for detection. High producers are isolated foractivity assay as described below.

20 high producers that express functionally active Glucocerebrosidaseare selected to be adapted into protein-free suspension culture in 125ml culture flasks. (HyQ PF media). Once adapted, the activity of theexpressed protein is ascertained again.

The top five clones are cultured in 2 L bioreactors to characterizetheir productivity and the activity of the expressed protein

Example 12 Glucocerebrosidase Activity Assay

The activity assay for Glucocerebrosidase activity uses afluorescence-based assay with 4-methylumberlliferyl β-D-glucopyranoside(4 MU β-D-Glu) as a substrate.

4 MU β-D-Glu is converted to 4-methylumberlliferone (4-MU) and glucosein presence of Glucocerebrosidase. Concentrations of 4 MU are used todetermine activity of glucocerebrosidase present in supernatant.

Supernatant samples are spun down to remove debris cells and transferredto clean centrifuge tubes. Samples are serially diluted 7 times withdeionized MilliQ water to obtain 500 uL of each 1×, 2×, 4×, 8×, 16×,32×, 64×, 128× diluted concentrations.

50 μL of each concentration of each sample loaded into 8 wells on 96well plate. Loaded wells are topped up to 100 μL with 500 μL 2× AssayBuffer supplemented with 1600 ug/mL (2×) 4-methylumberlliferylβ-D-glucopyranoside (4 MU β-D-Glu) and incubated at 37° C. in a staticincubator.

Enzymatic reactions in 2 wells of each concentration of each samplequenched with 100 uL of Stop Buffer at 15 min, 2 h, 4 h and 6 hrespectively. Wells scanned for fluorescence using Tecan SPECTRAFluorPlus with excitation wavelength 360+/−10 nm and emission wavelength440+/−10 nm

Concentrations of 4 MU present in quenched samples compared to standardcurve of 4 MU.

Standard curve of 4 MU prepared by serially diluting 1 M of 4 MU in DMSOwith 1× Assay Buffer to obtain concentrations of 1× (1 mM), 2×, 4×, 8×,16×, 32×, 64×, 128×, 256×, 512×, 1024×, 2048×, 4096×, 8192×, 16 k× and32 k×

100 μL of each concentration of 4 MU sample loaded into 2 wells andtopped to 200 μL with 100 μL of Stop Buffer.

Example 13 Purification of Expressed Glucocerebrosidase

Glucocerebrosidase is purified according to modified protocol publishedin Proc. Natl. Acad. Sci 1977, 74, No. 8, 3560-3563.

A decyl-agarose column (length-to-diameter ratio, 5:1) is packed andequilibrated with 0.1 M sodium citrate buffer, pH 5.0/1 mM2-mercaptoethanol/5 mM EDTA. This buffer is used in the following stepsand will be referred to hereafter as citrate buffer.

The dialyzed butanol extract is clarified by centrifugation at 5000×gfor 30 min, and the supernatant is applied to the column at a ratio of50,000 units/ml of column volume. The column is then washed withone-half column volume of citrate buffer followed by elution with an 8column-volume linear gradient of 30-80% ethylene glycol in citratebuffer.

Glucocerebrosidase is eluted on the trailing edge of a protein peak atan ethylene glycol concentration of approximately 60%. Fractions withhigh specific activity are pooled and diluted 1:3 with citrate buffer.

An octyl-Sepharose column (length-to-diameter ratio, 5:1) is packed,washed for 12 hr with 0.1 M NaOH, and equilibrated with citrate bufferprepared in pyrogen-free 0.9% (wt/vol) saline. The enzyme sample isapplied at a ratio of 300,000 units/ml of column volume. The column iswashed with one-half column volume of pyrogen-free citrate buffer.

Approximately-40% of the inactive protein did not adsorb to the column,and the enzyme is eluted at about 60% ethylene glycol concentration.

The fractions from the octyl-Sepharose column with high specificactivity are pooled, diluted by one-third with 60% ethylene glycol in0.9% saline, and made 0.5 mg/ml in human serum albumin. This enzymesolution is added rapidly to three volumes of cold 95% ethanol, stirredwell, and held in the cold for 1 hr before centrifugation at 4000×g for5 min.

The supernatant is discarded and the precipitate is suspended in 200volumes (wt/vol) of an ethanol/glycerol solution (three volumes of 95%ethanol and one volume of 60% pyrogen-free glycerol); the suspension iscentrifuged as above. The wash is repeated twice more and the finalprecipitate is taken up in human serum albumin solution (40 mg/ml in0.9% saline) to a concentration of approximately 106 units/ml.

Example 14 Mass Spectrometry of N-glycans Produced by JW152

EPO-Fc was expressed in wild type CHO-K1 cells and JW152 cells andpurified by affinity chromatography. N-glycans were released frompurified EPO-Fc and analyzed by Mass spectrometry (MS).

The results are shown in FIG. 7. Mass spectrometry results of N-glycansproduced by wild-type CHO-K1 cells are shown on the top panel and thoseproduced by JW152 cells are shown at the bottom panel.

As can be seen, CHO-K1 cells produced all kinds of N-glycans. However,JW152 cells produced only mannose-terminated N-glycans: Man5GlcNAc2,fucosylated Man5GlcNAc2 and Man4GlcNAc.

Example 15 Comparison of Glucocerebrosidase Activity in Supernatant ofTransfected Cells and Untransfected Cells

Culture supernatant is incubated with 4-methylumbelliferyl®-D-glucopyranoside in a citrate buffer (pH 5.9).

After one hour, the reaction is quenched with addition of 0.2M glycineadjusted to pH 10.5.

4-methylumbelliferone (4-MU) gives peak fluorescence activity above pH10. Fluorescence is measured at λex 365 nm; λm 445 nm.

A standard curve is generated from dilution of 4-MU reagent. Theactivity is based on measured amount of 4-MU released in one hour. WhenGlucocerebrosidase was expressed in JW152 cells, the higher activity wasobserved in the culture medium.

References

References 1 to 10 are for the section headed “Gaucher's Disease” above.

1. James, William D.; Berger, Timothy G.; et al. (2006). Andrews'Diseases of the Skin: clinical Dermatology. Saunders Elsevier. ISBN0-7216-2921-0.

2. a b Jacquelyn K Beals (Nov. 19, 2008), “ASHG 2008: Gaucher DiseaseMutation Carriers at Higher Risk for Parkinson's Disease”, MedscapeMedical News

3. a b Gaucher PCE (1882). De l'epithelioma primitif de la rate,hypertrophie idiopathique de la rate sans leucemie [academic thesis].Paris, France.

4. Aharon-Peretz J, Rosenbaum H, Gershoni-Baruch R (2004). “Mutations inthe glucocerebrosidase gene and Parkinson's disease in Ashkenazi Jews”.N. Engl. J. Med. 351 (19): 1972-7. doi:10.1056/NEJMoa033277. PMID15525722.

5. Landgren O, Turesson I, Gridley G, Caporaso N E (2007). “Risk ofMalignant Disease Among 1525 Adult Male US Veterans With GaucherDisease”. Archives of Internal Medicine 167 (11): 1189-1194.doi:10.1001/archinte.167.11.1189. PMID 17563029.

6. Online ‘Mendelian Inheritance in Man’ (OMIM) 606463

-   7. a b Grabowski G A (2008). “Phenotype, diagnosis, and treatment of    Gaucher's disease”. Lancet 372: 1263-1271.    doi:10.1016/S0140-6736(08)61522-6.

8. Weinreb N J, Deegan P, Kacena K A, et al. (December 2008). “Lifeexpectancy in Gaucher disease type 1”. Am. J. Hematol. 83 (12): 896-900.doi:10.1002/ajh.21305. PMID 18980271.

9. Diaz G A, Gelb B D, Risch N, et al. (2000). “Gaucher disease: theorigins of the Ashkenazi Jewish N370S and 84GG acid beta-glucosidasemutations”. Am. J. Hum. Genet. 66 (6): 1821-32. doi:10.1086/302946. PMID10777718.

10. www.medicalnewstoday.com/articles/180630.php

11. “National Gaucher Foundation”. Retrieved 2007 May 30.

12. Brady R O, Kanfer J N, Shapiro D (1965). “Metabolism ofglucocerebrosides. II. Evidence of an enzymatic deficiency in Gaucher'sdisease”. Biochem. Biophys. Res. Commun. 18: 221-5.doi:10.1016/0006-291X(65)90743-6. PMID 14282020.

13. geneticpeople.com/?p=276

14. www.nlm.nih.gov/medlineplus/ency/imagepages/1450.htm

15. Dvir et al., EMBO Reports, 2003, 4:704

16. Helenius & Aebi, Science, 2001, 291:2364

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A recombinant protein comprising mannose-terminated N-glycans expressed by a host cell line according to a method which includes steps of: (a) introducing a nucleic acid encoding a recombinant protein into a host cell comprising a mutation in the GnT I gene leading to loss of GnT I function, wherein the host cell is a Chinese Hamster Ovary (CHO) cell selected with Ricinus communis agglutinin I (RCA-I) or a descendent thereof; and (b) expressing the recombinant protein from the host cell, wherein the expressed recombinant protein comprises mannose-terminated N-glycans and the method does not include a step of introducing functional GnT I into the host cell.
 2. The recombinant protein comprising mannose-terminated N-glycans according to claim 1, wherein the recombinant protein has glucocerebrosidase activity.
 3. The recombinant protein comprising mannose-terminated N-glycans according to claim 1, wherein the recombinant protein is MUC1.
 4. The recombinant protein comprising mannose-terminated N-glycans according to claim 1, wherein the recombinant protein is HER2/neu.
 5. The recombinant protein comprising mannose-terminated N-glycans according to claim 1, wherein the recombinant protein is carcinoembryonic antigen (CEA).
 6. A method comprising expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell according to claim 1, and administering the recombinant protein comprising mannose-terminated N-glycans to an individual suffering from or at risk of suffering from: a cancer and the recombinant protein comprising mannose-terminated N-glycans is a tumor antigen; Gaucher's disease and the recombinant protein comprising mannose-terminated N-glycans has glucocerebrosidase activity; a viral associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a viral antigen; a bacterial associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a bacterial antigen; a parasite associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a parasitic antigen; or an allergic disease and the recombinant protein comprising mannose-terminated N-glycans is an allergen.
 7. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is MUC1, and the individual is suffering from or at risk of suffering from a cancer.
 8. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is HER2/neu, and the individual is suffering from or at risk of suffering from a cancer.
 9. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is carcinoembryonic antigen (CEA), and the individual is suffering from or at risk of suffering from a cancer.
 10. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans has glucocerebrosidase activity, and the individual is suffering from Gaucher's disease.
 11. A method comprising administering a recombinant protein comprising mannose-terminated N-glycans according to claim 1 to an individual suffering from or at risk of suffering from: a cancer and the recombinant protein comprising mannose-terminated N-glycans is a tumor antigen; Gaucher's disease and the recombinant protein comprising mannose-terminated N-glycans has glucocerebrosidase activity; a viral associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a viral antigen; a bacterial associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a bacterial antigen; a parasite associated or caused disease and the recombinant protein comprising mannose-terminated N-glycans is a parasitic antigen; or an allergic disease and the recombinant protein comprising mannose-terminated N-glycans is an allergen.
 12. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is MUC1 and the individual is suffering from or at risk of suffering from a cancer.
 13. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is HER2/neu, and the individual is suffering from or at risk of suffering from a cancer.
 14. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is carcinoembryonic antigen (CEA), and the individual is suffering from or at risk of suffering from a cancer.
 15. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans has glucocerebrosidase activity, and the individual is suffering from Gaucher's disease.
 16. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is a viral antigen selected from the group consisting of antigens from hepatitis viruses A, B, C, D & E, HIV, herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus, and measles virus, and the individual is suffering from a viral associated or caused disease.
 17. The method according to claim 16, wherein the viral antigen is selected from the group consisting of HIV-1 antigens tat, nef, gp120 or gp160, gp40, p24, gag, env, vif, vpr, vpu, rev or part and/or combinations thereof; human herpes virus antigens gH, gL, gM, gB, gC, gK, gE, gD, or part and/or combinations thereof, Immediate Early proteins ICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2; cytomegalovirus antigens gB or derivatives thereof; Epstein Barr virus antigens gp350 or derivatives thereof; Varicella Zoster Virus antigens asgp1, 11, 111 and IE63; hepatitis virus antigens env protein E1, env protein E2, core protein, NS2, NS3, NS4a, NS4b, NSSa, NSSb, p7, or part and/or combinations thereof; human papilloma virus antigens L1, L2, E1, E2, E3, E4, E5, E6, E7, or part and/or combinations thereof; Respiratory Syncytial virus antigens F and G proteins or derivatives thereof; parainfluenza virus antigens; measles virus antigens; mumps virus antigens; flavivirus antigens; and Influenza virus antigens HA, NP, NA, M proteins, or part and/or combinations thereof, and the individual is suffering from a viral associated or caused disease.
 18. The method according to claim 16, wherein the viral antigen is selected from the group consisting of Human herpesvirus 5 envelope glycoprotein B and Human herpesvirus 4 (Epstein-Barr virus) glycoprotein 350/220, and the individual is suffering from a viral associated or caused disease.
 19. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is a bacterial antigen selected from the group consisting of antigens from Mycobacteria causing TB and leprosy, pneumocci, aerobic gram negative bacilli, mycoplasma, bacteria causing staphyloccocal infections, bacteria causing streptococcal infections, bacteria causing salmonellae, bacteria causing chlamydiae, and bacteria causing neisseriae, and the individual is suffering from a bacterial associated or caused disease.
 20. The method according to claim 6, wherein the recombinant protein comprising mannose-terminated N-glycans is a parasitic antigen selected from the group consisting of antigens from parasites causing malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, and filariasis, and the individual is suffering from a parasite associated or caused disease.
 21. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is a viral antigen selected from the group consisting of antigens from hepatitis viruses A, B, C, D & E, HIV, herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus, and measles virus, and the individual is suffering from a viral associated or caused disease.
 22. The method according to claim 21, wherein the viral antigen is selected from the group consisting of HIV-1 antigens tat, nef, gp120 or gp160, gp40, p24, gag, env, vif, vpr, vpu, rev or part and/or combinations thereof; human herpes virus antigens gH, gL, gM, gB, gC, gK, gE, gD, or part and/or combinations thereof, Immediate Early proteins ICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2; cytomegalovirus antigens gB or derivatives thereof; Epstein Barr virus antigens gp350 or derivatives thereof; Varicella Zoster Virus antigens asgp1, 11, 111 and IE63; hepatitis virus antigens env protein E1, env protein E2, core protein, NS2, NS3, NS4a, NS4b, NSSa, NSSb, p7, or part and/or combinations thereof; human papilloma virus antigens L1, L2, E1, E2, E3, E4, E5, E6, E7, or part and/or combinations thereof; Respiratory Syncytial virus antigens F and G proteins or derivatives thereof; parainfluenza virus antigens; measles virus antigens; mumps virus antigens; flavivirus antigens; and Influenza virus antigens HA, NP, NA, M proteins, or part and/or combinations thereof, and the individual is suffering from a viral associated or caused disease.
 23. The method according to claim 21, wherein the viral antigen is selected from the group consisting of Human herpesvirus 5 envelope glycoprotein B and Human herpesvirus 4 (Epstein-Barr virus) glycoprotein 350/220, and the individual is suffering from a viral associated or caused disease.
 24. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is a bacterial antigen selected from the group consisting of antigens from Mycobacteria causing TB and leprosy, pneumocci, aerobic gram negative bacilli, mycoplasma, bacteria causing staphyloccocal infections, bacteria causing streptococcal infections, bacteria causing salmonellae, bacteria causing chlamydiae, and bacteria causing neisseriae, and the individual is suffering from a bacterial associated or caused disease.
 25. The method according to claim 11, wherein the recombinant protein comprising mannose-terminated N-glycans is a parasitic antigen selected from the group consisting of antigens from parasites causing malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, and filariasis, and the individual is suffering from a parasite associated or caused disease. 